Composition and method to alleviate joint pain using low molecular weight hyaluronic acid and astaxanthin

ABSTRACT

A dietary supplement composition is formulated in a therapeutic amount to treat and alleviate symptoms of joint pain. The composition includes pro-inflammatory low molecular weight microbial fermented sodium hyaluronate fragments having a molecular weight of 0.5 to 300 kilodaltons (kDa) in an oral dosage form.

PRIORITY APPLICATION(S)

This is a divisional of application Ser. No. 15/194,957 filed Jun. 28,2016 (now U.S. Pat. No. 9,999,631), which is a continuation ofapplication Ser. No. 14/735,370 filed Jun. 10, 2015 (now U.S. Pat. No.9,675,635), which is a continuation of application Ser. No. 14/645,805filed Mar. 12, 2015 (now U.S. Pat. No. 9,402,857), which is acontinuation-in-part application of Ser. No. 14/217,515 filed Mar. 18,2014 (now U.S. Pat. No. 9,238,043), which is a continuation-in-partapplication of Ser. No. 13/914,725 filed Jun. 11, 2013 (now U.S. Pat.No. 8,945,608), which is a continuation application of Ser. No.12/840,372 filed Jul. 21, 2010 (now U.S. Pat. No. 8,481,072), which isbased upon provisional application Ser. No. 61/227,872 filed Jul. 23,2009; and provisional application Ser. No. 61/345,652 filed May 18,2010, the disclosures which are hereby incorporated by reference intheir entirety.

FIELD OF THE INVENTION

This invention relates to treating and alleviating joint pain andsymptoms of osteoarthritis and/or rheumatoid arthritis.

BACKGROUND OF THE INVENTION

The use of krill oil is disclosed in U.S. Patent Publication Nos.2004/0234587; 2004/0241249; and 2007/0098808, the disclosures which arehereby incorporated by reference in their entirety. The use of krill oilis also disclosed in a research paper published by L. Deutsch entitled,“Evaluation of the Effect of Neptune Krill Oil on Chronic Inflammationand Arthritic Symptoms,” published in the Journal of the AmericanCollege of Nutrition, Volume 26, No. 1, 39-49 (2007), the disclosurewhich is hereby incorporated by reference in its entirety.

The published '587, '249 and '808 applications discuss the beneficialaspects of using krill oil in association with pharmaceuticallyacceptable carriers. As an example, this krill and/or marine oil can beobtained by the combination of detailed steps as taught in the '808application, by placing krill and/or marine material in a ketonesolvent, separating the liquid and solid contents, recovering a firstlipid rich fraction from the liquid contents by evaporation, placing thesolid contents and organic solvent in an organic solvent of the type astaught in the specification, separating the liquid and solid contents,recovering a second lipid rich fraction by evaporation of the solventfrom the liquid contents and recovering the solid contents. Theresultant krill oil extract has also been used in an attempt to decreaselipid profiles in patients with hyperlipidemia. The '808 publicationgives details regarding this krill oil as derived using those generalsteps identified above.

SUMMARY OF THE INVENTION

A dietary supplement composition is formulated in a therapeutic amountto treat and alleviate symptoms of joint pain. The composition includespro-inflammatory low molecular weight microbial fermented sodiumhyaluronate fragments and glucosamine in an oral dosage form.

In one example, the pro-inflammatory, low molecular weight microbialfermented sodium hyaluronate fragments may have a molecular weight of0.5 to 300 kilodaltons (kDa). In another example, the pro-inflammatorylow molecular weight microbial fermented sodium hyaluronate fragmentsare micro- or nano-dispersed within the composition. The glucosamine maybe formed as glucosamine hydrochloride (HCL) or glucosamine sulfate. Thecomposition may include at least one of chondroitin, Bowellia, turmeric,methylsulfonymethane (MSM), and s-adenosyl-methionine.

In another example, a dietary supplement composition is formulated in atherapeutic amount to treat and alleviate symptoms of joint pain. Thecomposition includes pro-inflammatory low molecular weight microbialfermented sodium hyaluronate fragments, glucosamine and Type II collagenin an oral dosage form.

A method to treat and alleviate symptoms of joint pain in a patientincludes administering a therapeutic amount of a dietary supplementcomposition comprising pro-inflammatory low molecular weight microbialfermented sodium hyaluronate fragments and glucosamine in an oral dosageform.

In another example, a method to treat and alleviate symptoms of jointpain in a patient includes administering a therapeutic amount of adietary supplement composition comprising pro-inflammatory low molecularweight microbial fermented sodium hyaluronate fragments, glucosamine andType II collagen in an oral dosage form.

DESCRIPTION OF THE DRAWINGS

Other objects, features and advantages of the present invention willbecome apparent from the detailed description of the invention whichfollows, when considered in light of the accompanying drawings in which:

FIG. 1 is a view showing a chemical structure of astaxanthin that can beused in accordance with a non-limiting example.

DETAILED DESCRIPTION

The present invention will now be described more fully hereinafter withreference to the accompanying drawings, in which preferred embodimentsof the invention are shown. This invention may, however, be embodied inmany different forms and should not be construed as limited to theembodiments set forth herein. Rather, these embodiments are provided sothat this disclosure will be thorough and complete, and will fullyconvey the scope of the invention to those skilled in the art.

There now follows a description of the joint health composition andassociated method as set forth in the '072, '608, and '275 patentsrelated to the krill oil and/or fish derived oil and includes noveldetails of an algae based oil, fish oil derived products, roe and/orplant based oils, including phospholipids, which are more removed fromthe omega-3 platform base. Novel details and new uses and compositionfrom different hyaluronic acid sources and phospholipids and astaxanthinare described.

The composition as related to the krill oil includes EPA and DHAfunctionalized as marine phospholipids and acyltriglycerides derivedfrom krill. The krill, algae, roe extract and fish oil derived productand phospholipid compositions may include astaxanthin, such asesterified astaxanthin, and in one non-limiting example, low molecularweight polymers of hyaluronic acid or sodium hyaluronate (hyaluronan) inan oral dosage form. In one example, it includes pro-inflammatory lowmolecular weight microbial fermented sodium hyaluronate having amolecular weight of between 0.5 to 300 kDa, in another example between0.5 to 230 kDa, and in yet another example, between 0.5 to 100 kDa. Someof these components relative to the krill oil in an example areexplained in the following chart:

Components Percentage (%) PHOSPHOLIPIDS PC, PE, PI, PS, SM, CL >40OMEGA-3 (functionalized on PL) >30 Eicosapentaenoid Acid (EPA)* >17 (15%in one example and 10% in another) Docosahexaenoid Acid (DHA) + >11 (9%in one example and 5% in another) ANTIOXIDANTS (mg/100 g) Astaxanthin,Vitamin A, Vitamin E >1.25 * >55% of PL-EPA/Total EPA + >55% ofPL-DHA/Total DHA These amounts can vary depending on application andpersons.

The composition includes pro-inflammatory microbial fermented sodiumhyaluronate fragments having a molecular weight of 0.5 to 300kilodaltons (kDa), in an example, and 0.5 to 230 kDa, and 0.5 to 100kDa, all in an oral dosage form. Natural high molecular weighthyaluronic acid is the major hydrodynamic component of synovial fluidand importantly is known to be immuno-neutral to the innate immunesystem. It is nature's bone joint shock absorbent and lubricant. It hasbeen found that there is excellent oral bioavailability of low molecularweight hyaluronic acid (LMWtHA) fragments specifically to connectivetissue, which maximizes interaction with target synovial fluid producingcells. Therefore in a preferred composition containing krill oil, algaebased oil, fish oil derived product, roe, and phospholipids or othercompositions, the astaxanthin and LMWtHA, two anti-inflammatorycomponents are thus combined with one highly inflammatory component.

The scientific literature indicates that LMWtHA fragments exhibit potentpro-inflammatory behavior. It therefore remains unclear why apro-inflammatory component would elicit a favorable overall response ininflamed joint tissues. It is believed that such pro-inflammatory LMWtHAfragments promote site repair by simulation of the innate immune systemrepair mechanism and by simulating production of non-immunogenic highmolecular weight hyaluronic acid bringing the joint back to homeostasis.A great deal of work by leading immunologists is still attempting tounravel all the aspects of the complicated signaling processesassociated with the innate immune system. Studies using large animalmodels of osteoarthritis have shown that mild immunogenic HyaluronicAcids with molecular weights within the range of 0.5-1.0×10⁶ Da (Dalton)were generally more effective in reducing indices of synovialinflammation and restoring the rheological properties of SF(visco-induction) than non-immunogenic HA's with molecular weights>2.3×10⁶ Da.

Those skilled in the art understand that pro-inflammatory low molecularweight hyaluronic acid is around 300 kDa to about 320 kDa or less, withmany skilled in the art using 300 kDa as the cut-off. Low molecularweight hyaluronic acids and sodium hyaluronates are well known to act aspro-inflammatory agents and assumed up-regulators of the inflammatorycascade with respect to the innate immune system. Some reports indicatethat hyaluronic acid fragments induce expression of inflammatory genesand they are low molecular weight kDa. Clinical trials by the inventorsand their assignee have shown the effectiveness of the composition whenusing krill oil, together with the low molecular weight hyaluronic acidor hyaluronan and astaxanthin in accordance with a non-limiting example.In the clinical trials, no rescue medication was allowed as compared tothe Deutsch study referenced above. The low molecular weight hyaluronicacid had a molecular weight of about 40 kDa in the trial, but couldrange from 0.5 to 100 kDa in an example, or 0.5 to 230 kDa, or 0.5 to300 kDa in yet other examples.

The composition and method used in the clinical trials of the currentsubject matter were directed to treating and alleviating joint pain. Theclinical subjects in the clinical trial did not have any confirmedosteoarthritis and/or rheumatoid arthritis. An abbreviated exclusioncriteria listed specifically that subjects did not have any presence ofauto-immune diseases or similar diseases and the study had excludedthose subjects who knew their joint pain was due to osteoarthritisand/or rheumatoid arthritis. The clinical study was directed to patientsthat have a non-disease state joint pain that is not associated with adisease state such as osteoarthritis and/or rheumatoid arthritis. Thecomposition was used as a supplement to treat and alleviate symptoms ofjoint pain of unknown etiology, including joint pain not associated withosteoarthritis and/or rheumatoid arthritis in this example.

Astaxanthin is a component of the composition. The clinical trials ofthe joint care composition with the krill oil, low molecular weighthyaluronic acid and astaxanthin proved the effectiveness of thecomposition with surprising beneficial results. Related scientificliterature indicates that in a lipopolysaccharide induced inflammatoryrat model, astaxanthin at just 1 mg/kg in vitro and in vivo: (1) downregulates TNF-alpha production by 75%; (2) down regulates prostaglandinE-2 production (PGE-2) by 75%; (3) inhibits nitric oxide synthase (NOS)expression of nitric oxide by 58%; and (4) these effects on inflammatorymarkers were nearly as effective as prednisolone in this model. Suchinformation suggests but does not prove that astaxanthin may be aneffective standalone product for the reduction of OA and/or RH pain orother symptomology associated with OA and/or RH. FIG. 1 shows an exampleof the astaxanthin as astaxanthin 3S, 3′S (3, 3′-dihydroxy-4,4′-diketo-β-carotene). The clinical trial of 15 mg astaxanthin alone isnoted as beneficial.

The incorporated by reference '072 and '608 patents describe thatclinical trial using astaxanthin alone where a dosage of one softgelcontaining 15 mg of astaxanthin as prepared and described was given oncea day during breakfast for 12 weeks. This large dosage of astaxanthinalone was effective to treat osteoarthritis and joint pain. It has nowbeen determined that lower dosages of astaxanthin may be used instead ofthese much higher dosages such as 15 mg in the clinical trial when it isadded with at least one of a phospholipid, glycolipid, and sphingolipidor used alone with the low molecular weight hyaluronic acid. Apharmaceutical or food grade diluent may be added or other surfactant.Other beneficial and often synergistic results are obtained whenastaxanthin is used in the presence of the low molecular weighthyaluronic acid as described above or UC-II. Phospholipids may includeplant based phospholipids such as from lecithin and lysophospholipidsand/or glycophospholipids, including perilla oil such as described incommonly assigned U.S. Pat. No. 8,784,904, the disclosure which ishereby incorporated by reference in its entirety. Astaxanthin levelscould very from 0.5-2 mg and 0.5-4 mg and in one embodiment is 2-4 mg or2-6 mg and as broad as 0.5-12 mg and 7-12 mg.

In induced uveitis, astaxanthin also showed dose dependant ocularanti-inflammatory activity by suppression of NO, PGE-2 and TNF-Alpha bydirectly blocking NO synthase activity. Astaxanthin is also known toreduce C-Reactive Protein (C-RP) blood levels in vivo. For example, inhuman subjects with high risk levels of C-RP three months of astaxanthintreatment resulted in 43% of patients serum C-RP levels to drop belowthe risk level. This may explain why C-RP levels dropped significantlyin the Deutsch study identified above. Astaxanthin is so powerful thatit has been shown to negate the pro-oxidant activity of Vioxx, a COX-2inhibitor belonging to the NSAIDS drug class which is known to causecellular membrane lipid peroxidation leading to heart attack and stroke.For this reason Vioxx was removed from the US market. Astaxanthin isabsorbed in vitro by lens epithelial cells where it suppresses UVBinduced lipid peroxidative mediated cell damage at umol/Lconcentrations. In human trials astaxanthin at 4 mgs/day prevented postexercise joint fatigue following strenuous knee exercise when comparedto untreated subjects. These results have been shown in:

-   1) Lee et al., Molecules and Cells, 16(1):97-105; 2003;-   2) Ohgami et al., Investigative Ophthalmology and Visual Science    44(6):2694-2701, 2003;-   3) Spiller et al., J. of the Amer. College of Nutrition, 21(5):    October 2002; and-   4) Fry et al., Univ. of Memphis Human Performance Laboratories, 2001    and 2004, Reports 1 & 2.

A composition in one embodiment includes 300 mg of krill oil, 30 to 45mg of low molecular weight hyaluronic acid, and 2 mg astaxanthin. It hasnow been found that 150 mg to 300 mg of krill oil is beneficial with oneembodiment using 150 mg. The astaxanthin can range from 0.5 to 2 mg, 2to 4 mg, 0.5 to 6 mg, 0.5 to 8 mg, 0.5 to 10 mg, 0.5 to 12 mg, and 7 to12 mg. The use of added phospholipids and/or surfactants described belowwill aid in delivery of the astaxanthin. The low molecular weighthyaluronic acid can vary from 10 to 70 mg, from 20 to 60 mg, from 25 to50 mg, with one embodiment having 45 mg, and in another embodiment about30 mg.

Astaxanthin has potent singlet oxygen quenching activity. Astaxanthintypically does not exhibit pro-oxidant activity unlike β-carotene,lutein, zeaxanthin and Vitamins A and E. Astaxanthin in some studies hasbeen found to be about 50 times more powerful than Vitamin E, 11 timesmore powerful than β-carotene and three times more powerful than luteinin quenching of singlet oxygen. Astaxanthin is also well known for itsability to quench free radicals. Comparative studies have foundastaxanthin to be 65 times more powerful than Vitamin C, 54 times morepowerful than β-carotene, 47 times more powerful than lutein, and 14times more powerful than Vitamin E in free radical quenching ability.

U.S. Pat. No. 5,527,533 (the Tso patent), the disclosure which is herebyincorporated by reference in its entirety, discloses the benefits ofastaxanthin for retarding and ameliorating central nervous system andeye damage. Astaxanthin crosses the blood-brain-retina barrier and thiscan be measured by direct measurement of retinal astaxanthinconcentrations. Thus, Tso demonstrated protection from photon induceddamage of photo-receptors, ganglion and neuronal cell damage.

Studies have shown that HA binds to the surface of dendritic cells(“DC's”) and stimulated T-cells. Blockade of the CD44-HA interactionleads to impaired T-Cell activation both in vitro and in vivo. Studieshave shown that in cancer cell lines, LMWtHA fragments specificallyinduce nitric oxide synthase in dendritic cells. In DC's, NO expressioncaused dendritic cell apoptosis (cell death). DC's are essential T-cellactivators which function by presenting antigens to T-cells, thusapoptosis of DC's may short circuit the adaptive immune system response.This effect was clearly CD44 dependent because pretreatment of DC's withanti-CD44 monoclonal antibodies blocked the NO mediated induction of DCapoptosis. It appears that low molecular weight HA fragments interruptthe normal course of the well known T-cell mediated adaptive immunesystem response. CD44 is a glycoprotein responsible in part forlymphocyte activation (also known as T-cell activation) and is known tospecifically bind to HA. On the other hand as previously discussed lowmolecular weight HA fragments appear to up-regulate the innate immuneresponse, particularly in chronic inflammatory conditions where theinnate immune system may in some way be compromised.

Support for such teachings can be found in:

-   1) Mummert et al., J. of Immunol. 169, 4322-4331;-   2) Termeer et al., Trends in Immunology, Vol. 24, March 2003;-   3) Yang et al., Cancer Res. 62, 2583-2591; and-   4) McKee et al., J. Biol. Chem. 272, 8013-8018.

Additional information can be found in the following references: GhoshP. Guidolin D. Semin Arthritis Rheum., 2002 August; 32(1):10-37; and P.Rooney, M. Wang, P. Kumar and S. Kumar, Journal of Cell Science 105,213-218 (1993).

As noted before, krill oil is typically produced from Antarctic krill(Euphausia superba), which is a zooplankton (base of food chain). It isone of the most abundant marine biomass of about 500 million tonsaccording to some estimates. Antarctic krill breeds in the pureuncontaminated deep sea waters. It is a non-exploited marine biomass andthe catch per year is less than or equal to about 0.02% according tosome estimates. Because krill is harvested in large amounts and worldsupply of krill is being depleted, substitutes for krill such as othermarine based oils, including algae based oils, are now being studied,developed and used.

It is believed that krill oil and some other marine based and plantbased oils have an oil based phospholipid bound EPA and DHA uptake intocellular membranes that is far more efficient than triacylglyercidebound EPA and DHA, since liver conversion of triacylglycerides is itselfinefficient and because phospholipid bound EPA and DHA can betransported into the blood stream via the lympathic system, thus,avoiding liver breakdown. In addition, krill, algae and some marine andplant based oil consumption does not produce the burp-back observed withfish oil based products. Because of this burp-back feature of fish oils,it has been found that approximately 50% of all consumers who try fishoil never buy it again. Some algae based oils have EPA conjugated withphospholipid and glycolipid polar lipids, making the EPA uptake evenmore efficient.

As to astaxanthin, it has an excellent safety record. A conducted studyobtained the results as follows:

Oral LD 50: 600 mg/kg (rats);

NOAEL: 465 mg/kg (rats); or

Serum Pharmacokinetics: Stewart et al. 2008

1) T_(1/2): 16 hours;

2) T_(max): 8 hours;

3) C_(max): 65 μg/L.

At eight weeks of supplementation at 6 mg per day, there was no negativeeffect in healthy adults. Spiller et al. 2003.

In accordance with one non-limiting example, astaxanthin has three primesources: 3 mg astaxanthin per 240 g serving of non-farmed raised salmonor a 1% to 12% astaxanthin oleoresin or 1.5-2.5% beadlet derived frommicroalgae. Further verification is reflected in Lee et al., Moleculesand Cells 16(1): 97-105, 2003; Ohgami et al., InvestigativeOphthalmology and Visual Science 44(6): 2694-2701, 2003; Spiller et al.,J. of the American College of Nutrition 21(5): October 2002; and Fry etal., University of Memphis, Human Performance Laboratories, 2001 and2004, Reports 1 and 2.

Beneficial and synergistic effects are now being reported herein andhave been observed when krill, algae, fish oil derived product, roeextract, and seed oil and other phospholipid based compositions are usedin combination with other active ingredients. More particularly, thecurrent composition has krill, algae, fish oil derived, roe, seed oil,or other phospholipid ingredients in combination with astaxanthin andlow molecular weight polymers of hyaluronic acid or sodium hyaluronatein preferably an oral dosage form for the control of joint pain range ofmotion and stiffness. It should be understood that different proportionsof the composition components and their percentages can be useddepending on end use applications and other environmental andphysiological factors when treating a patient.

In accordance with a non-limiting example, the composition and methodtreats and alleviates symptoms of non-disease state joint pain and maybe used to treat and alleviate symptoms of osteoarthritis and/orrheumatoid arthritis in a patient by administering a therapeutic amountof the composition, including the krill oil or other algae based oil,fish oil derived product, roe, and other phospholipid materials incombination with astaxanthin and low molecular weight polymers ofhyaluronic acid or sodium hyaluronate (hyaluronan) in an oral dosageform, preferably the low molecular weight polymers. The krill oil alone,in one example, is derived from Euphasia spp., comprisingEicosapentaenoic (EPA) and Docosahexaenoic (DHA) fatty acids in the formof triacylglycerides and phospholipids, although not less than 1% EPAand 5% DHA has been found advantageous.

In another example, the krill oil includes at least 15% EPA and 9% DHA,of which not less than 45% are in the form of phospholipids, and inanother example 40%. The composition can be delivered advantageously fortherapeutic results with 1-4000 mg of oil, such as krill or algae basedoil, delivered per daily dose. In another example, 500 mg is a preferredamount for a single capsule dosage, and in another example 1,000 mg. Inanother example, 0.1-50 mg astaxanthin are supplemented to the oil perdaily dose, but a preferred amount is about 2-4 mg and 0.5 to 12 mg. Thealgae and other marine based oils and roe extract with phospholipid andplant based oils and phospholipids may be used. The composition of thealgae based oils and their fatty acid profile varies from the fatty acidprofiles of krill oil as explained below and shown in the tables. It ispossible to also use wax esters and omega-3 salts and ethyl esters.

The composition may also include an n-3 (omega-3) fatty acid rich oilderived from fish oil, algae oil, flax seed oil, or chia seed oil whenthe n-3 fatty acid comprises alpha-linolenic, stearidonic,eicosapentaenoic or docosapentaenoic acid. The composition may includenaturally-derived and synthetic antioxidants that are added to retarddegradation of fatty acids and astaxanthin.

Details of a type of CO2 extraction and processing technology (assupercritical CO2 extraction) and peroxidation blocker technology thatcan be used are disclosed in commonly assigned U.S. Pat. Nos. 8,652,544;8,586,104; 8,784,904; and U.S. Patent Publication No. 2009/0181114, thedisclosures which are hereby incorporated by reference in theirentirety.

As noted before, there are beneficial aspects of using krill oil oralgae based oil and other oils as described in synergistic combinationwith other ingredients. It has been determined that a fish oil derived,choline based, phospholipid bound omega-3 fatty acid mixture includingphospholipid bound polyunsaturated EPA and DHA is advantageous for jointhealth when combined with the astaxanthin and low molecular weighthyaluronic acid or hyaluronate. One commercially available example of amixture of fish oil derived, choline based, phospholipid bound fattyacid mixture including polyunsaturated EPA and DHA is Omega Choline1520F as a phospholipid, omega-3 preparation, which is derived fromnatural fish oil and sold by Enzymotec Ltd. One example of suchcomposition is described below:

Ingredients (g/100 g): Pure Marine Phospholipids n.l.t. 15 DHA* n.l.t.12 EPA** n.l.t. 7 *Docosahexaenoic acid **Eicosapenteanoic acid Omega-3n.l.t. 22 Omega-6 <3 Analytical Data: Peroxide value (meq/Kg) n.m.t. 5Loss on Drying (g/100 g) n.m.t. 2 Physical Properties: ConsistencyViscous Liquid

The mixture of fish oil derived, choline based, phospholipid bound fattyacid mixture including polyunsaturated EPA and DHA in one examplecomprises Eicosapentaenoic (EPA) and Docosahexaenoic (DHA) fatty acidsin the form of triacylglycerides and phospholipids. In another example,the omega choline includes at least 7% EPA and 12% DHA, of which notless than 15% are in the form of phospholipids. The composition can bedelivered advantageously for therapeutic results with 1-4000 mg of amixture of fish oil and fish oil derived, choline based, phospholipidbound fatty acid mixture including polyunsaturated EPA and DHA deliveredper daily dose. In one example, about 150 mg to about 300 mg is used. Inanother example, 2 to 4 mg astaxanthin are supplemented to the omegacholine per daily dose, but may include a range of 0.5 to 4 mg, or 0.5to 6 mg, 0.5 to 12 mg, or 7 to 12 mg, and other ranges as describedbefore.

It is also possible to use a mixture of fish oil derived, choline based,phospholipid bound omega-3 fatty acid mixture (including polyunsaturatedEPA and DHA) mixed with astaxanthin and the low molecular weighthyaluronic acid. It should also be understood that an enriched versionof a mixture of fish oil derived, choline based, phospholipid boundfatty acid mixture including polyunsaturated EPA and DHA can be usedwherein the fraction of added fish oil diluents has been decreased andthe proportion of fish oil derived phospholipids has been increased.This can be accomplished by using supercritical CO2 and/or solventextractions for selective removal of triacylglycerides fromphospholipids such as using the techniques in the incorporated byreference patents. The composition may also include a natural orsynthetic cyclooxygenase-1 or -2 inhibitor comprising for exampleaspirin, acetaminophen, steroids, prednisone, or NSAIDs. The compositionmay also include a gamma-linoleic acid rich oil comprising Borage(Borago officinalis L.) or Safflower (Carthamus tinctorius L.), whichdelivers a metabolic precursor to PGE₁ synthesis.

The composition may also include an n-3 (omega-3) fatty acid rich oilderived from fish oil, algae oil, flax seed oil, chia seed oil orperilla seed oil wherein the n-3 fatty acid source comprisesalpha-linolenic, stearidonic, eicosapentaenoic or docosapentaenoic acid.The composition may include naturally-derived and synthetic antioxidantsthat are added to retard degradation of fatty acids such as tocopherols,tocotrienols, carnosic acid or Carnosol and/or astaxanthin.

It has been found advantageous to use herring roe extract as the sourceof phospholipids that may have some EPA and DHA. Synergistic results areobtained and vast improvements seen. One study indicated thatphospholipids from herring roe improved phospholipid and glucosetolerance in healthy, young adults as published by Bjorndal et al.,Lipids in Health Disease, 2014, 13:82. The pure roe phospholipid may beformed using extraction techniques. It is a honey-like product that isthinned or diluted with fish oil and/or perilla oil or other seed orplant oil, in an example.

The specification prior to dilution with fish oil and/or perilla oil isas follows:

Percentage that is phospholipids 60 Phospholipid mg/g 600 Phosphatidylcholine portion mg/g 520 Choline equivalents 83 Total EPA mg/g (TG & PLbound) 75 Total DHA mg/g (TG & PL bound) 195 EPA mg/g bound tophospholipid 67 DHA mg/g bound to phospholipid 175 EPA + DHA mg/g boundto phospholipid 242

The herring roe extract is processed in one example using extraction byethanol. Triacylglycerides are added and ethanol stripped out to have arobust solution. Seed oil, such as the perilla seed oil as described inthe incorporated by reference '904 patent, may be added back to theethanol extract before stripping to thin and form a high levelphospholipid blend. The roe oil extract may be mixed with fish oiland/or seed oil, such as the perilla, or any other marine oil. In anexample, the herring egg roe extract is mixed with perilla seed oil ofat least 1:1 and preferably as high as 6:1 ALA to LA with theconcentrate as having at least 50%, and in another example 60%phospholipids, and in another example at least 30%, and in anotherexample 40% triglycerides.

An example composition includes a combination of a roe extract fromherring or a phospholipid rich roe extract with phospholipid bound EPAand DHA admixed with seed/fish oil and/or seed oil where the seed oilhas a ratio of ALA to LA between 1:1 and 1:6, and optionally includingastaxanthin in one example of about 2-4 mg or 0.5 to 12 mg or otherranges as noted above, and the low molecular weight hyaluronic acid,such as described above. The amount of roe egg extract mixed with theseed oil such as perilla oil varies and is about 150 to 500 mg, or 300to 500 mg, or up to 1,000 mg daily dose in one example and may includehyaluronic acid. Other plant based phospholipids may be used, includingcommercially available lecithins and an egg yolk derivative, includinglysophospholipids and glycophospholipids to act as surfactants. It ispossible to use sunflower-based phospholipids and natural plant-basedoils and natural surfactant extracts. The astaxanthin is enhanced withfats, surfactants, or phospholipids and can be delivered moreefficiently with phospholipids and sunflower based and/or the lipophilicperilla oil as described before.

In an example, the perilla oil is formed as a shelf stable,supercritical, CO2 fluid extracted seed oil derived from a crackedbiomass of Perilla frutescens from 60 to 95 percent w/w of PUFAs in aratio of from 4:1 to 6:1 alpha-linolenic acid (ALA) to linoleic acid(LA). The Perilla frutescens derived seed oil is made in an example bysubjecting the Perilla frutescens seed to supercritical fluid CO2extraction to produce a seed oil extract; fractionating the resultingseed oil extract in separate pressure step-down stages for collectinglight and heavy fractions of seed oil extract; and separating the heavyfraction from the light fraction to form the final seed oil from theheavy fraction.

Selected antioxidants are included in another example and the perillaoil includes a mixture of selected lipophilic and hydrophilicantioxidants. Lipophilic antioxidants can be used either alone or incombination with at least one of: a) phenolic antioxidants including atleast one of sage, oregano, and rosemary; b) tocopherol; c)tocotrienol(s); d) carotenoids including at least one of astaxanthin,lutein, and zeaxanthin; e) ascorbylacetate; f) ascorbylpalmitate; g)Butylated hydroxytoluene (BHT); h) Docosapentaenoic Acid (BHA); or i)Tertiary Butyl hydroquinone (TBHQ). A hydrophilic antioxidant orsequesterant may include hydrophilic phenolic antioxidants including atleast one of grape seed extract, tea extracts, ascorbic acid, citricacid, tartaric acid, and malic acid.

In one example, a peroxide value of this perilla seed oil is under 10.0meq/Km. In another example, this perilla seed oil is from 85 to 95percent w/w of PUFAs and the PUFAs are at least greater than 56 percentalpha-linolenic acid (ALA). The perilla seed oil is shelf stable at roomtemperature up to 32 months. In another example, this perilla seed oilis derived from a premilled or flake-rolled cracked biomass of Perillafrutescens. The mixture of selected antioxidants may includeastaxanthin, phenolic antioxidants and natural tocopherols. The perillaseed oil may also include at least one of dispersed nano- andmicro-particles of rice or sugar cane based policosanol.

In an example, the composition is encapsulated into a single dosagecapsule and referred to as a deep ocean caviar capsule. In a specificexample, the encapsulated composition includes herring caviarphospholipid extract (herring roe) perilla (Perilla frutescens) seedextract, olive oil, Zanthin® astaxanthin (Haematococcus pluvialis algaeextract), gelatin, spice extract, non-GMO natural tocopherols,cholecalciferol, riboflavin, and methylcobalamin. The compositionincludes fish as herring roe and tilapia gelatin. An example is setforth in the following chart.

Properties: Appearance Size 00 clear capsule with dark red oily fillFatty Acids ALA min. 140 mg EPA min. 18 mg DHA min. 50 mg Total Omega-3min. 210 mg Phospholipids 195 mg Astaxanthin 500 μg Vitamin D₃ 1000 IU;250% DV Vitamin B₂ (Riboflavin) 1.7 mg; 100% DV Vitamin B₁₂ 6 μg; 100%DV Microbiological USP <61>/FDA SAM Total Plate Count <1000 cfu/g Yeast& Mold <100 cfu/g E. coli Absent in 10 g Salmonella Absent in 10 g S.aureus Absent in 10 g Storage Conditions Tightly closed containers,15-30° C., 30-50% RH Shelf-life 24 months minimum Packaging HDPE or PETbottle (count TBD) All ingredients BSE-free and non-GMO

The processing components may contain a mix of marine omega-3phospholipids derived from herring caviar and perilla seed oil. It maycontain an O2B™ botanical peroxidation blocker, including spice extract,non-GMO tocopherols and ascorbyl palmitate. It can be packaged as a bulkproduct in sealed drums 45 and 190 kg net with inert headspace,complying with European and American standards for food products. Itpreferably stores at below room temperature. The product is protectedagainst light and heat. If drums are opened for sampling, the headspacecan be flushed with inert gas during sampling and prior to storing.

Test Unit Acceptance Criterion Method Appearance Amber viscous oilAM2020 Solubility Oil soluble and AM2021 water dispersible MinimumMaximum ALA (C18:3 n-3) mg/g as TG³⁾ 230 AM1044 EPA (C20:5 n-3) mg/g asTG³⁾ 30 AM1001 DHA (C22:6 n-3) mg/g as TG³⁾ 85 AM1001 Total omega-3 ¹⁾mg/g as TG³⁾ 370 AM1001 ALA (C18:3 n-3) mg/g as FFA⁴⁾ 215 AM1044 EPA(C20:5 n-3) mg/g as FFA⁴⁾ 28 AM1001 DHA (C22:6 n-3) mg/g as FFA⁴⁾ 80AM1001 Total omega-3¹⁾ mg/g as FFA⁴⁾ 335 AM1001 Total PC mg/g 250 AM1002Total PL mg/g 300 AM1002 Total neutral lipids mg/g 700 AM1003 Watercontent by % 3.0 AM1004 Karl Fisher Peroxide value meq/kg 10.0 AM1005Heavy metals (sum mg/kg 10 AM1015 of Pb, Hg, Cd & In-organic As) ²⁾ ¹⁾Total n-3: ALA, EPA, DHA, 18:4, 20:4, 21:5, 22:5 ²⁾ Frequency analysis³⁾ All ALA, EPA, DNA or Total omega-3 expressed as triglycerides ⁴⁾ AllALA, EPA, DHA or Total omega-3 expressed as free fatty acids

It has been surprisingly found that the astaxanthin may be made morebioavailable when incorporated or used with one of at least aphospholipid, glycolipid, and sphingolipid and optionally with foodand/or pharmaceutical grade diluents. Lower dosages as compared to the15 mg used in previous clinical trials may be used. The astaxanthin isat least about 0.1 to about 15 percent by weight of the at least onephospholipid, glycolipid, and sphingolipid. The astaxanthin in anexample is derived from a natural or synthetic ester or synthetic diol.A pharmaceutical or food grade diluent may be added. When incorporatedwith a microbial fermented, low molecular weight hyaluronic acid orsodium hyaluronate (hyaluronan) as described before, a dietarysupplement composition is formed and can be formulated in a therapeuticamount to treat and alleviate symptoms of joint pain in a person havingjoint pain.

It should be understood that the triglycerides have two types ofmolecules as a glycerol and three fatty acids, while the phospholipidscontain glycerol and fatty acids, but have one glycerol molecule and twofatty acid molecules. In place of that third fatty acid, a polar groupis instead attached to the glycerol molecule so that the phospholipidsare partly hydrophilic as compared to hydrophobic triglycerides.Lysophospholipids may be used as a derivative of a phospholipid in whichone or both acyl derivatives have been removed by hydrolysis. Lecithinand its derivatives may be used as an emulsifier and surfactant as awetting agent to reduce surface tension of liquids. Other phospholipidsmay be used. Different phospholipids include phosphatidylcholine,phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol,phosphatidic acid, lyso-phosphatidylcholine,lyso-phosphatidylethanolamine, and lyso-Phosphatidylserine. Some may bederived from egg yolk and extracted chemically using hexane, ethanol,acetone, petroleum ether or benzene, and also extracted mechanically,including from different sources such as soybeans, eggs, milk, marinesources, and sunflower. When derived from soya and sunflower,phospholipids may include those products mentioned before, includingphosphatidic acid. Various compositions such as lecithin may behydrolyzed enzymatically and have a fatty acid removed by phospholipaseto form the lysophospholipids that can be added to the roe extract asexplained above. One phospholipase is phospholipase A2 where the fattyacid is removed at the C2 position of glycerol. Fractionation may beused.

The glycolipids are primarily derivatives of ceramides where a fattyacid is bonded or connected to the amino alcohol sphingosine. It shouldbe understood that the phospholipid sphingomyelin is also derived from aceramide. Glycolipids, however, contain no phosphates in comparison tothe phospholipids. The fat is connected to a sugar molecule in aglycolipid and are fats bonded to sugars. Because it is built from asphingosine, fat and sugar, some refer to it as a glycosphingolipid. Asphingolipid is a lipid that contains a backbone of sphingoid basis andset of alphatic amino alcohols that include the sphingosine. As notedbefore, the phospholipid and other components may be derived from atleast one of a plant, algae and animal source, or a synthetic derivativethereof. The phospholipid and other components may be derived from atleast one of soybean, sunflower, grapeseed, egg yolk, krill, fish body,fish roe, squid, and algae. The phospholipid and other components may beformed as compound rich mono- or di-glcerides or fatty acids where thefatty acid contains between 2 and 20 carbon atoms. During processing,the composition is formed by dispersing the astaxanthin and phospholipidand optionally a diluent under high shear conditions. The diluent may bea pharmaceutical or food grade diluent as known to those skilled in theart.

In another example, the astaxanthin is about 2 to about 10 percent byweight of the phospholipid and glycolipid and derived from a natural orsynthetic ester or synthetic diol. In yet another example, 50 to 500 mgof phospholipid, glycolipid, and sphingolipid may be used. The dietarysupplement composition may be formulated into a single dosage capsule.

The astaxanthin may be derived from Haematococcus pluvialis algae,Pfaffia, krill, or by synthetic routes, in the free or synthetic diol,monoester or diester form, both natural and synthetic, at a daily doseof 0.5-8 mg or 0.5-12 mg, in one example, and in another example, 1-2mg, 2-4 mg, 1-6 mg, and other ranges, and up to 12 mg, including 7-12mg. The polymers of hyaluronic acid or sodium hyaluronate (hyaluronan)can be derived from microbial fermentation or animal tissue. About 1-500mg of hyaluronan can be delivered per daily dose and preferably between10 and 70 mgs/dose and at 20 to 60, 25 to 50, and 35 and 45 mg per dose.The hyaluronan is micro- or nano-dispersed within the composition in onepreferred example. In another example, the hyaluronic acid is derivedfrom a biofermenation process and has a molecular weight between 0.5 and100 kilodaltons (kDa), and in another example, up to 300 kDa andpreferably 0.5 to 300 kDa, and in another example, from 0.5 to 230 kDaas low molecular weight hyaluronic acid or hyaluronan. A preferred rangeis 0.5 to 300 kDa. In another example, the polymers of hyaluronic acidor sodium hyaluronate (hyaluronan) are derived from microbialfermentation or animal tissue.

The pure low molecular weight hyaluronic acid oligomers in an exampleare derived principally and practically from microbial fermentation, butcould also be derived from hydrolyzed animal tissues. This microbialfermentation process is known to produce extraordinarily pure lowmolecular sodium hyaluronate free from amino acid conjugation.

Human hyaluronic acid is typically synthesized in the body naturally ortaken from the diet such as from chicken, beef, and other naturalsources. This natural hyaluronic acid has high molecular weight, i.e.,greater than 300 kDa, as compared to microbial fermented sodiumhyaluronate that is low molecular weight and defined in the literatureas about 0.5 to 300 kDa. The hyaluronic acid naturally found in the bodyis a polymer of acidified glucuronic acid and N-acetyl-glucosamine,which under physiological pH of about 7.4, exists as free acid, withpartial sodium, potassium and ammonium salts. Streptococcus in oneexample is used to ferment the sodium hyaluronate and is a mutantstrain. Therefore, the resulting low molecular weight hyaluronic acid isobtained from a mutant strain of streptococcus bacteria. Thefermentation process is followed by isolation and denaturation of theorganism and its proteins with ethanol and heat. This is followed byfiltration. The molecular weight is chemically modified with acidaqueous chemical hydrolysis as a chemical reaction. The final product isisolated by ethanol precipitation of the sodium salt and drying toproduce pro-inflammatory low molecular weight microbially fermentedsodium hyaluronate fragments.

This low molecular weight sodium hyaluronate is a chemical reactiondegradation product of a mutant strain streptococcus bacterialfermentation. An example sodium hyaluronate is manufactured byfermentation using the bacterial strain Streptococcus zooepidemicus. Theproduction strain is a non-hemolytic mutant of a parent strain, NCTC7023. The production strain is produced by nitroso-guanidine mutagenesiswith a unique ribosomal genome sequence not naturally found in nature.

This manufacturing process has three main stages of 1) fermentation, 2)purification, and 3) refining. The fermentation begins with a seedculture from the mutant production strain. A starter culture inoculatesthe seed tank, which contains a broth medium that is grown out to becomethe seed broth. The seed broth is transferred to a fermenter containingthe sterilized culture medium and a culturing temperature of 33-37degrees Celsius is maintained until fermentation is complete within22-30 hours.

This fermentation broth is mixed with ethanol to obtain precipitated,crude sodium hyaluronate. The 50-70% ethanol concentration used duringpurification inactivates the streptococcus organism. The crude productis dissolved in purified water and filtered to remove both impuritiesand inactivated microbial fragments. This yields a clear filtrate. Thewater has a temperature of 50-70 degrees Celsius when used in thedissolution step and inactivates any remaining streptococcus organism.The target molecular weight sodium hyaluronate is then obtained bycontrolling the pH, temperature and holding time in the dissolutionstep. The higher the pH and temperature in the specified range, and thelonger the holding time in the specified range, the lower the resultingmolecular weight of the sodium hyaluronate will be. The filtratecontaining the chemical hydrolysis derived low molecular weighthyaluronic acid produced during the chemical molecular weightmodification step is then precipitated with ethanol, followed by washingor dehydrating. The precipitate is dried under vacuum to yield the finallow molecular weight, microbial fermented sodium hyaluronate.

Other sources for low molecular weight hyaluronic acid may be used.These include low molecular weight hyaluronic acid derived from chickensternal cartilage extract. The hyaluronic acid may include elastin,elastin precursors, and collagen. The hyaluronic acid may be containedin a matrix form with chondroitin sulfate and naturally occurringhydrolyzed collagen Type II nutraceutical ingredients and form lowerweight molecules that the body may more readily absorb and deliver todifferent areas of the body as required. Fresh chicken sternal cartilagecould be cut and suspended in aqueous solution followed by treating thecartilage with a proteolytic enzyme to form a hydrolysate. Theproteolytic enzyme is capable of hydrolyzing collagen Type II tofragments having a lower molecular weight. The hydrolysate is sterilizedand filtered and concentrated and then dried to form powder enrichedcollagen Type II powder that is then isolated and includes a percentageof low molecular weight hyaluronic acid. Examples of manufacturingtechniques can be found in U.S. Pat. Nos. 6,780,841 and 6,025,327, thedisclosures which are hereby incorporated by reference in theirentirety.

It is possible that the low molecular weight hyaluronic acid could alsobe derived from the hydrolyzed collagen as derived from the bovinecollagen Type I or the chicken sternal cartilage collagen Type II, oreven a natural eggshell membrane that includes some hyaluronic acid,which can be extracted from the eggshell membrane. Although someteachings will take the hyaluronic acid derived from eggshell membranesuch as in the incorporated by reference patents, the hyaluronic acid isprocessed to increase its molecular weight using cross-linkingtechniques as compared to using a low molecular weight hyaluronic acid.The eggshell membrane can still be used to obtain the low molecularweight hyaluronic acid. It may be possible to use enzymatic degradationof eggshell membrane that undergoes manipulation to purify thehyaluronic acid.

The hyaluronic acid may be derived from dehydrated rooster combs such asdisclosed in U.S. Pat. No. 6,806,259 and U.S. Patent Publication No.2006/0183709, which are incorporated herein by reference in theirentirety, where the hyaluronic acid may be further processed. Often itis a higher molecular weight and will be processed to obtain a lowermolecular weight of the desired 0.5 to 300 kDa. In many teachings, acertain molecular weight hyaluronic acid is processed to increase itsmolecular weight. The hyaluronic acid may also be obtained from humanumbilical cords or other techniques such as disclosed in U.S. Pat. No.4,141,973, the disclosure which is hereby incorporated by reference inits entirety, and further processed to obtain the desired molecularweight.

It has been determined that synergistic or advantageous improvements canbe made to some commercially available compositions that include about50 mg of an active ingredient, for example, hyaluronic acid and acartilage, such as a Type II collagen when astaxanthin is added.Sometimes boron is used. For example, the composition includes 30-50 mgof collagen and about 4-6 mg of boron and 2-4 mg of hyaluronic acid withan average of each of the component ranges. It has been found that aneffective and synergistic result is obtained when astaxanthin is addedalone and/or low molecular weight hyaluronic acid such as 0.5 to 4 mg or0.5 to 12 mg of astaxanthin plus 30-45 mg of low molecular weighthyaluronic acid, although even smaller amounts could be used, such as1-5 mg. This composition could include Type II collagen with the addedastaxanthin and low molecular weight hyaluronic acid with the optionaladdition of boron. One (1) to 500 mg of hyaluronic acid could be used.

In an example, a cartilage blend as a mixture of cartilage and salt isabout 40 mg with boron as 5 mg and hyaluronic acid as 3.3 mg. Thecartilage blend includes cartilage and potassium chloride to provide 10mg of undenatured Type-2 collagen. It is possible for anothercomposition to include the astaxanthin with the composition that isformed from glucosamine hydrochloride such as about 1.25 to 1.75 orabout 1.5 grams and methylsulfonymethane (MSM) of about 500 to 1,000 andabout 750 mg and including the addition of chondroitin sulfate of about150 to 250 and about 200 mg. It also may include the joint fluid ashyaluronic acid, such as 1-5 mg and about 3.3 mg, and also vitamin D3and other components such as antioxidants. The astaxanthin can varybetween 2 to 4 mg or 0.5 to 12 mg and other ranges as disclosed above.It should be understood that the astaxanthin and the at least one ofphospholipid, glycolipid, and sphingolipid or other components asdescribed above may be used for many different purposes and results. Itmay be used to aid in treating or improving blood lipid profiles andreducing LDL per-oxidation in humans. It may be used to counter or treatdepression and other neurological disorders. It may be used forrespiratory illnesses and skin ailments or diseases.

It has been found advantageous and synergistic to use astaxanthin withlow molecular weight hyaluronic acid. It can be incorporated optionallywith the UC-II with ranges as described above. Astaxanthin beadletscould be added to the UC-II. This type of composition is advantageousover glucosamine chondroitin pills that require two much larger pills aday to support joint and cartilage. The composition may include anatural or synthetic cyclooxygenase-1 or -2 inhibitor comprising forexample aspirin, acetaminophen, steroids, prednisone, or NSAIDs. Thecomposition may also include a gamma-linoleic acid rich oil comprisingBorage (Borago officinalis L.) or Safflower (Carthamus tinctorius L.),which delivers a metabolic precursor to PGE₁ synthesis.

The composition may also include an n-3 (omega-3) fatty acid rich oilderived from fish oil, algae oil, flax seed oil, chia seed oil, orperilla seed oil. In an example, the n-3 fatty acid comprisesalpha-linolenic, stearidonic, eicosapentaenoic or docosapentaenoic acid.In one example composition as noted before, it has been found that analgae based oil may be used instead of krill oil. Hydrolyzed orunhydrolyzed collagen and elastin derived from eggshell membranes canalso be advantageously added. The composition may also includeanti-inflammatory and/or natural joint health promoting compoundscomprising at least one of preparations of green lipped mussel (Pernacanaliculus), Boswellia serrata, turmeric (Curcuma longa), stingingnettle (Urtica dioica), Andrographis, Cat's claw (Uncaria tomentosa),bromelain, methylsulfonylmethane (MSM), chondroitin sulfate, glucosaminesulfate, s-adenosyl-methionine, proanthocyanidins, procyanidins orflavonoids. The composition may include naturally-derived and syntheticantioxidants that are added to retard degradation of fatty acids andastaxanthin.

Different compositions may use different ingredients in combination withthe krill, algae or other oil, including the seed based oil, roeextract, and phospholipid and other surfactants. The astaxanthin andhyaluronate may be combined with different ingredients and supplementalcompositions for more specific purposes.

A pharmaceutically acceptable composition comprises a krill, fish,algae, roe extract or plant based oil and/or phospholipid and/orsurfactant in combination with astaxanthin and hyaluronate optionallycombined with one or more ingredients including but not limited toglucosamine sulfate, chondroitin sulfate, collagen, methylsulfonmethane,a gamma-linoleic acid or omega-3 fatty acid rich oil a cyclooxgenaseinhibitor or a lipogenase inhibitor for the treatment of symptomsrelated to non-disease joint pain and/or joint diseases, including butnot limited to osteoarthritis and rheumatoid arthritis.

In yet another example, a dietary supplement acceptable compositioncomprises a krill, algae, fish, roe extract, or plant based oil and/orother phospholipid and/or surfactant in combination with astaxanthin andhyaluronate optionally combined one or more ingredients, including butnot limited to, glucosamine sulfate, chondroitin sulfate, collagen,methylsulfonmethane, a gamma-linoleic acid or omega-3 fatty acid richoil a cyclooxgenase inhibitor or a lipoxygenase inhibitor for thetreatment of symptoms related to non-disease joint pain and/or jointdiseases, including but not limited to osteoarthritis and rheumatoidarthritis.

In yet another example, a medical food acceptable composition comprisesa krill, algae, fish, roe extract, or plant based oil and/or otherphospholipid and/or surfactant in combination with astaxanthin andhyaluronate and optionally combined with one or more ingredientsincluding glucosamine sulfate, chondroitin sulfate, collagen,methylsulfonmethane, a gamma-linoleic acid or omega-3 fatty acid richoil, a cyclooxgenase inhibitor or a lipoxygenase inhibitor for thetreatment of symptoms related to non-disease joint pain and/or jointdiseases, including but not limited to osteoarthritis and rheumatoidarthritis.

In still another example, a composition is formulated in a therapeuticamount to treat and alleviate symptoms of non-disease joint pain and/orjoint diseases, including osteoarthritis and/or rheumatoid arthritis,wherein the composition includes a krill, algae, fish, roe extract, orplant based oil and/or other phospholipid and/or surfactant incombination with astaxanthin and polymers of hyaluronic acid or sodiumhyaluronate (hyaluronan) in an oral dosage form. This compositionincludes other active constituents as explained and identified aboverelative to the method and composition.

The composition oil, whether from krill, algae, fish, roe extract, orplant based oil, and/or other phospholipid and/or surfactant, is usedwith the HA, such as the low molecular weight HA, and astaxanthin totreat non-disease joint pain in one example, but can be used to treatosteoarthritis. Osteoarthritis (OA) is the most prevalent form ofarthritis and is a disease in which the cartilage that acts as a cushionbetween the bones in joints begins to wear away causing bone on bonejoint swelling and joint pain. It is characterized by degeneration ofarticular cartilage along with peri-articular bone response. It affectsboth sexes, mainly in the fourth and fifth decades of life. The kneejoint is most commonly affected joint. At present the management is bypharmacological and non-pharmacological therapy. Corrective surgicaltherapy and or joint replacement therapy in some cases may not bepossible.

Traditional treatments for osteoarthritis involve the use of analgesics,non-steroidal anti-inflammatory drugs (NSAIDs) or cyclooxygenase-2specific (COX-2) NSAIDs alone or in combination. Advances in recombinantprotein synthesis also provide relief from the symptoms of OA and RH.Steroid or high molecular weight hyaluronic acid injections have alsobeen used with some success however these therapies have well knowndeleterious side effects.

Many of these treatments alone have shown limited effectiveness inclinical trials. To avoid the cardiac risks and gastrointestinal issuesassociated with traditional OA treatments (particularly with long termuse), many patients have turned to complimentary and alternativemedicines (CAMs) such as dietary supplements. Glucosamine andchondroitin alone or in combination, are widely marketed as dietarysupplements to treat joint pain due to OA. Two major clinical trials onglucosamine and chondroitin (The GAIT Study) failed to show anysignificant improvement in WOMAC score over placebo except in thehighest quartile of patients studied. Because of their limitedeffectiveness, the search for additional CAMs to treat OA continues (seefor example Ruff et al., Eggshell Membrane in the Treatment of Pain andStiffness from Osteoarthritis of the Knee: A Randomized, Multicenter,Double-Blind, Placebo-Controlled Clinical Study, Clin. Rheumatol (2009)28:907-914).

It is also possible to use a pure diol of the S, S′ astaxanthin,including a synthetic diol with a surfactant and/or the low molecularweight hyaluronic acid. It is possible to use that pure diol incombination with the EPA rich algae based oil or other fish, roeextract, or plant based oil and/or phospholipid and/or surfactant asdescribed above, and which is admixed with either astaxanthin derivedfrom Haematococcus pluvialis or the free diol form in substantially pureS,S′ enantiomer form. It is possible to add synthetically derived mixedenantiomers of the diol. The diol of the S, S′ astaxanthin is possiblebecause in cases of krill oil and possibly algae based oils and Hpderived and other types, there are principally diesters and monoestersrespectively with very little diol, which is insoluble. Some researchindicates that it may be many times more bioavailable than either themonoester or diester form. It is possible to synthesize asymmetricallythe S,S′ pure diol. Despite the pure diol's poor solubility in someexamples, there may be an active transport mechanism related to itsbioavailability, or conversely, that only in the diol form is themonoester or diester forms transferred from the intestines to the blood.The phospholipid or glycolipid based product presenting EPA and/or DHAalong with the added astaxanthin in its various forms and especially theS,S′ enantiomeric form in principally monoester form from Haematococcuspluvialis or pure diol form from asymmetric synthesis could be viable.Thus, it is possible to combine it with the algae derived glycol andphospholipid based EPA rich oil.

As noted before, astaxanthin (3,3′-dihydroxy-β-β-carotene-4,4′-dione) isa xanthophyll carotenoid found in many marine species includingcrustaceans, salmonid fish and algae. Astaxanthin cannot be synthesizedby mammals, but when consumed in the diet has shown effectiveness as anantioxidant, anti-inflammatory agent and with benefit to eye health,heart health, and the immune system.

Astaxanthin has a hydroxyl group on each β-ionone moiety, therefore itcan be found in its free (diol) form as well as mono- or di-esterified.In natural products astaxanthin is commonly found as a mixture:primarily mono-esters of C12-C18 fatty acids and lesser amounts ofdi-ester and free diol. Synthetic astaxanthin is commonly provided inonly the free diol form.

The astaxanthin molecule has two E/Z chiral centers and three opticalR/S isomers. Haematococcus pluvialis algae produces natural astaxanthinsolely in the (3S,3′S) isomer. This is explained in the article fromRenstrøm B., G. Borch, O. Skulberg and S. Liaane-Jensen, “Optical Purityof (3S,3′S) Astaxanthin From Haematococcus Pluvialis,” Phytochemistry,20(11): 2561-2564, 1981, the disclosure which is hereby incorporated byreference in its entirety.

Alternatively, the yeast Phaffia rhodozyma synthesizes only the 3R,3′Rconfiguration. This is explained in the article from Andrewes A. and M.Starr entitled, “(3R,3′R)-Astaxanthin from the Yeast Phaffia Rhodozyma,”Phytochemistry, 15:1009-1011, 1976, the disclosure which is herebyincorporated by reference in its entirety.

Wild salmon predominately contain the (3S,3′S) form with a (3S,3′S),(3R,3′S), and (3R,3′R) isomer ratio of 22:1:5. This is explained in thearticle from Turujman, S, W. Warner, R. Wei and R. Albert entitled,“Rapid Liquid Chromatographic Method to Distinguish Wild Salmon FromAquacultured Salmon Fed Synthetic Astaxanthin,” J. AOAC Int., 80(3):622-632, 1997, the disclosure which is hereby incorporated by referencein its entirety.

However, astaxanthin produced by traditional synthesis will contain aracemic mixture in a (3S,3′S), (3R,3′S; meso), (3R,3′R) ratio of 1:2:1.This ratio is also seen in many species of shrimp, which are able toracemize (3S,3′S) to the meso form. This is explained in the articlefrom Schiedt, K., S. Bischof and E. Glinz entitled, “Metabolism ofCarotenoids and in vivo Racemization of (3S,3′S)-Astaxanthin in theCrustacean Penaeus,” Methods in Enzymology, 214:148-168, 1993, thedisclosure which is hereby incorporated by reference in its entirety.

However, most of the astaxanthin in shrimp is within the carapace(shell) therefore limited amounts of the meso isomer are consumed in thehuman diet.

Feeding studies of free diol or fatty acid esters of astaxanthin hasbeen shown to increase the amount of astaxanthin in human plasma. Thisare explained in the article from Osterlie, M., B. Bjerkeng and S.Liaan-Jensen, entitled “Plasma Appearance and Distribution ofAstaxanthin E/Z and R/S Isomers in Plasma Lipoproteins of Men AfterSingle Dose Administration of Astaxanthin,” J. Nutr. Biochem,11:482-490, 2000; and the article from Coral-Hinostroza, G., T.Ytestøyl, B. Ruyter and B. Bjerkeng entitled, “Plasma Appearance ofUnesterified Astaxanthin Geometrical E/Z and Optical R/S Isomers in MenGiven Single Doses of a Mixture of Optical 3 and 3′R/S Isomers ofAstaxanthin Fatty Acyl Diesters,” Comp. Biochem Phys. C., 139:99-110,2004, the disclosures which are hereby incorporated by reference intheir entirety.

The uptake of free astaxanthin diol is about 4-5 times higher than thatof esterified astaxanthin, likely due to the limitation of requiredenzymatic hydrolysis in the gut prior to absorption. These intestinalenzymes may also be R/S selective on astaxanthin esters.Coral-Hinostroza et al. (2004) found higher relative absorption ofastaxanthin from (3R,3′R-astaxanthin dipalmitate compared to the othertwo isomers. However, ingestion of racemic free diol astaxanthin doesnot show any stereospecific selection.

Astaxanthin for use in human food supplements is currently derived fromthe cultivated freshwater algae Haematococcus pluvialis. This algaeproduces 3S,3′S astaxanthin ester in a fatty acid matrix which can beisolated with solvent or carbon dioxide extraction. This oily extractcan be used directly in edible formulations or further processed intosolid powder or beadlet preparations. Many clinical studies have beenconducted with H. pluvialis derived astaxanthin to demonstratebeneficial health effects and safety. Food additive approvals forastaxanthin-rich algae extracts have been approved for many suppliers inthe US and EU.

Haematococcus algae cultivation for use in dietary supplements cannotalways match demand for use of astaxanthin in dietary supplements. Useof synthetic astaxanthin diol can also benefit applications which need aconcentrated, standardized astaxanthin source. Conventional racemicsynthetic astaxanthin sources are used as a colorant in Salmonidaquaculture as a feed ingredient. This racemic mixture may have limiteduse since only one-quarter of the compound is the 3S,3′S isomer commonlyfound in natural Salmon and has been studied in humans for efficacy andsafety.

Astaxanthin may also be synthesized with in a stereospecific manner, sothat the output is exclusively the generally accepted 3S,3′S isomer in afree diol form. The free diol crystals can be suspended in a vegetableoil or solid beadlet for use in edible preparations or pill, capsule,tablet form. The 3S,3′S product has the advantage of greater consistencythan algal preparations and also with lower odor. Thereforealgal-derived astaxanthin can be replaced with synthetic 3S,3′Sastaxanthin diol in existing formulations with the same or increasedeffectiveness.

As noted before, it has also been surprisingly found that the use ofhyaluronic acid alone and/or in combination with astaxanthin isbeneficial and synergistic. For example, low molecular weight hyaluronicacid in its different forms can be given to patients in an amount from1-500 mg per day and preferably about 10-70 mg per day, and in anotherexample, 20-60 mg, 25-50 mg, 35 mg, and 45 mg. Astaxanthin of about 2-4mg may be added in an example, but could range from 0.5 to 4 mg a day,and 7-12 mg range in another example, or 0.5 to 12 mg. The hyaluronicacid may be given in the form of a pro-inflammatory low molecular weightsodium hyaluronate fragments that are about 0.5-300 kDa corresponding tothe pro-inflammatory low molecular weight fragments. Although the use ofastaxanthin and phospholipids such as from krill oil, algae oil, roe,fish oil product, or plant based oils helps in delivering the hyaluronicacid, still the low molecular weight hyaluronic acid and in the form ofthe fragments preferably is still small enough to enter through the gutand be used in an oral administration.

It is also advantageous to use astaxanthin with the low molecular weighthyaluronic acid. Different amounts can be used, and in one example, 2-4mg per day, and in another example, 0.5-12 mg per day can be used withlow molecular weight hyaluronic acid such as the amount of 1-500 mg andpreferably about 10-70 mg and with 0.5-12 mg or 4-12 mg of astaxanthin.About 40-120 mg of low molecular weight hyaluronic acid may be used inan example. A dosage of astaxanthin may be about 6-8 mg and the lowmolecular weight hyaluronic acid could be in the range of about 60-80mg. Although the greater amounts of astaxanthin may be used with lowmolecular weight hyaluronic acid alone, it is possible to use 2 mg ofastaxanthin and lower amounts of low molecular weight hyaluronic acidsuch as 20 mg and up to 40 mg as non-limiting examples. It should beunderstood that hyaluronic acid fragments such as the pro-inflammatorylow molecular weight sodium hyaluronate fragments are potent as innateimmune system cell receptors signaling molecules associated with theinflammatory cascade and the oral hyaluronic acid in the form of lowmolecular weight fragments can reach joints as compared to the highermolecular weight hyaluronic acid that is injected since it is not orallyadministered.

As noted above, algae based oil having been found advantageous in anexample. This algae based oil provides an algae sourced EPA or anEPA/DHA based oil in which oils are present in phospholipid andglycerolipid forms, as glycolipids. Different algae based oils derivedfrom different microalgae may be used. One preferred example algae basedoil has the EPA titre higher than the DHA as compared to a class ofomega-3's from fish oils that are triacylglycerides. These algae basedoils are rich in EPA and in the phospholipid and glycolipid forms. Anexample marine based algae oil is produced by Parry Nutraceuticals as adivision of EID Parry (India) Ltd. as an omega-3 (EPA) oil.

It is known that algae can be an important source for omega-3 fattyacids such as EPA and DHA. It is known that fish and krill do notproduce omega-3 fatty acids but accumulate those fatty acids from thealgae they consume. Omega-3 bioavailability varies and is made availableat the site of physiological activity depending on what form it iscontained. For example, fish oil contains omega-3 fatty acids in atriglyceride form that are insoluble in water and require emulsificationby bile salts via the formation of micelles and subsequent digestion byenzymes and subsequent absorption. Those omega-3 fatty acids that arebound to polar lipids, such as phospholipids and glycolipids, however,are not dependent on bile for digestion and go through a simplerdigestion process before absorption. Thus, these omega-3 fatty acids,such as from an algae based oil, have greater bioavailability for cellgrowth and functioning as compared to the omega-3 triglycerides of fishoil. There are many varieties of algae that contain EPA conjugated withphospholipid and glycolipid polar lipids or contain EPA and DHAconjugated with phospholipids and glycolipids.

Throughout this description, the term “algae” or “microalgae” may beused interchangeably to each other with microalgae referring tophotosynthetic organisms that are native to aquatic or marine habitatsand are too small to be seen easily as individual organisms with thenaked eye. When the term “photoautotropic” is used, it refers to growthwith light as the primary source of energy and carbon dioxide as theprimary source of carbon. Other forms of biomass that may encompassalgae or microalgae may be used and the term “biomass” may refer to aliving or recently dead biological cellular material derived from plantsor animals. The term “polar” may refer to the compound that has portionsof negative and/or positive charges forming negative and/or positivepoles. The term “oil” may refer to a combination of fractionable lipidfractions of a biomass. As known to those skilled in the art, this mayinclude the entire range of various hydrocarbon soluble in non-polarsolvents and insoluble, or relatively insoluble in water as known tothose skilled in the art. The microalgae may also include any naturallyoccurring species or any genetically engineered microalgae to haveimproved lipid production.

The following first table shows the specification of an algae based oilas manufactured by Parry Nutraceuticals identified above, followed by asecond table for a fatty acid profile chart of that algae based oil. Athird table is a comparative chart of the fatty acid profiles fornon-algae based oils. These charts show that the algae based oil has ahigh EPA content of phospholipids and glycolipids.

Specification: Algae Based Oil

TEST SPECIFI- SOP. METHOD/ PARAMETERS CATION NO REFERENCE PhysicalProperties Appearance Viscous oil QA-88 In house Color Brownish QA-88 Inhouse black Odor Characteristic QA-88 In house Taste CharacteristicQA-88 In house General Composition Loss on drying (%)  2.0-3.0 QA-038USP <731> Loss on drying Ash (%)  0.5-1.0 QA-080 AOAC Official Method942.05, 16th Edition Protein (%)  1.0-2.0 QA-021 AOAC Official method978.04, 16th Edn. Carbohydrate (%)  1.0-2.0 AOAC 18th Edn 2006/ByDifference Residual Solvent (ppm) NMT 100 QA-074 GC-Head (as EthylAcetate) NMT 30 Space, (as Acetone) USP <467) Lipid Composition TotalLipid (%) 92.0-95.0 QA-86 AOAC official method 933.08 Chlorophyll (%)NMT 1.50 QA-078 Jeffrey & Humphrey (1975) Photosynthetic pigments ofAlgae (1989) Total carotenoids (%) NMT 1.50 QA-85 By JHFA method-1986Total Unsaponifiables (%) NMT 12.0 QA-086 AOAC official method 933.08Omega 3 [EPA + HDA] - NLT 15.00 QA-087 In House % w/w method Total Omega3 (% w/w) NLT 17.00 Total Omega 6 (% w/w) NMT 5.00 Total EFA (% w/w) NLT20. Lipid percentage Triglycerides 15-20% Phospholipids  5-10%Glycolipids 35-40% Free fatty acids 15-20% Microbial parameters StandardPlate Count NMT 1, 000 QA-039 AOAC, 1995, (cfu/1 g) Chapter 17 Yeast &Mold (cfu/1 g) NMT 100 Coli forms (/10 g) Negative E. Coli (/10 g)Negative Staphylococcus (/10 g) Negative Salmonella (/10 g) NegativeFatty acid profile (Area %) Myristic acid [14.0] NLT 4.0 QA-086 In HouseGC Palmiltic acid [16:0] NLT 16.0 & 087 method Palmito oleic acid NLT12.0 [16:1, n-9] Hexadecadienoic acid NLT 4.0 [16:2, n-4]Hexadecatrienoic acid NLT 12.0 [16:3, n-4] Stearic acid [18:0] NLT 0.10Oleic acid [18:1] NLT 1.0 Linoleic acid [18:2, n-6] - NLT 1.0 LA AlphaLinolenic acid NLT 0.50 [18:3, n-3] - ALA Stearidonic acid [18:4, NLT0.10 n-3] - SA Arachidonic Acid [20:4, NLT 0.25 n-6] - AAEicosapentaenoic acid NLT 15.0 [20:5, n-3] Decosahexaenoic acid NLT 1.5[20:6, n-3] Heavy Metals Lead (ppm) NMT 1.0 External AOAC 18th Arsenic(ppm) NMT 0.5 lab Edn: 2006 By Cadmium (ppm) NMT 0.05 reports ICPMSMercury (ppm) NMT 0.05 Safety: Safe for the intended use Shelf life: 24months from the date of manufacture Stability: Stable in unopenconditions Storage: Store in a cool, dry place away from sunlight, flushcontainer with Nitrogen after use Documentation: Every Batch of shipmentcarries COA Packing: 1 kg, 5 kg, and 20 kg food grade containers

Fatty Acid Profile Chart Algae Based Oil

ALGAE BASED OMEGA-3 FATTY ACID (EPA) OIL Total fatty acid, gm/100 gm ofoil 75 gm Fatty acid [% of total fatty acid] Myristic acid [14:0] 6.87Pentadecanoic acid [15:0] NA Palmitic acid [16:0] 20.12 Palmito oleicacid [16:1, ω-9] 18.75 Hexadecadienoic acid [16:2, ω-4] 6.84Hexadecatrienoic acid [16:4, ω-4] 12.54 Heptadecanoic acid [17:0] NAStearic acid [18:0] 0.68 Oleic acid [18:1, ω-9] 3.56 Linoleic acid[18:2, ω-6] 2.68 Alpha linolenic acid [18:3, ω-3] 3.73 Gamma linolenicacid [18:3, ω-6] NA Stearidoni acid [18:4, ω-3] 0.33 Arachidonic acid[20:4, ω-6] 0.97 Eicosapentaenoic acid [20:5, ω-3] EPA 23.00Docosapentaenoic acid [22:5, ω-3] DHA NA Docosahexaenoic acid [22:6,ω-3] DHA 3.26 Others 3.54 EPA/DHA [gm/100 gm oil] 15.75 Total ω-3 fattyacids [gm/100 gm oil] 18.20 LIPD CLASS DETAILS [gm/100 gm oil]Unsaponifiables [carotenoids, chlorophyll, 12 sterol, fatty alcoholetc.,] Free fatty acids 20 Triglycerides 20 Phospholipids 10 Glycolipids38 Total 100 STABILITY [months] 24

Fatty Acid Profile—Comparative Chart Non-Algae Based Oils

FISH OIL KRILL MARTEK FATTY ACID MAXEPA OIL OIL Total fatty acid, gm/100gm of oil 95 gm 70-80 gm 95 gm Fatty acid [% of total fatty acid]Myristic acid [14:0] 8.68 11.09 11.47 Pentadecanoic acid [15:0] NA NA NAPalmitic acid [16:0] 20.35 22.95 26.36 Palmito oleic acid [16:1, ω-9]11.25 6.63 NA Hexadecadienoic acid [16:2, ω-4] NA NA NA Hexadecatrienoicacid [16:4, ω-4] NA NA NA Heptadecanoic acid [17:0] NA NA NA Stearicacid [18:0] 4.67 1.02 0.50 Oleic acid [18:1, ω-9] 13.07 17.93 1.50Linoleic acid [18:2, ω-6] 1.28 0.14 0.61 Alpha linolenic acid [18:3,ω-3] 0.33 2.11 0.40 Gamma linolenic acid [18:3, ω-6] NA NA NAStearidonic acid [18:4, ω-3] 1.69 7.01 0.33 Arachidonic acid [20:4, ω-6]0.50 NA NA Eicosapentaenoic acid [20:5, ω-3] 20.31 19.04 1.0 EPADocosapentaenoic acid [22:5, ω-3] NA NA 15.21 DHA Docosahexaenoic acid[22:6, ω-3] 13.34 11.94 42.65 DHA others 4.53 0.14 NA EPA/DHA [gm/100 gmoil] 31.96 21.68 41.46 Total ω-3 fatty acids [gm/100 gm 33.85 28.0041.60 oil] LIPD CLASS DETAILS [gm/100 gm oil] Unsaponifiables[carotenoids, 5 5 5 chlorophyll, sterol, fatty alcohol etc.,] Free fattyacids 0.5 30 0.5 Triglycerides 94.5 25 94.5 Phospholipids Nil 40 NilGlycolipids Nil Nil Nil Total 100 100 100 STABILITY [months] 12 24 6

Different types of marine based algae oils may be used, includingNannochloropsis oculata as a source of EPA. Another algae that may beused is Thalassiosira weissflogii such as described in U.S. Pat. No.8,030,037 assigned to the above-mentioned Parry Nutraceuticals, aDivision of EID Parry (India) Ltd., the disclosure which is herebyincorporated by reference in its entirety. Other types of algae asdisclosed include chaetoceros sp. or prymnesiophyta or green algae suchas chlorophyta and other microalgae that are Diamons tiatoms. Thechlorophyta could be tetraselmis sp. and include prymnesiophyta such asthe class prymnesiophyceae and such as the order isochrysales and morespecifically, isochrysis sp. or pavlova sp.

There are many other algae species that can be used to produce EPA andDHA as an algae based oil whether marine based or not to be used inaccordance with a non-limiting example. In some cases, the isolation ofthe phospholipid and glycolipid bound EPA and DHA based oils may requiremanipulation of the algae species growth cycle.

Other algae/fungi phospholipid/glycolipid sources include: Grateloupiaturuturu; Porphyridium cruentum; Monodus subterraneus; Phaeodactylumtricornutum; Isochrysis galbana; navicula sp.; Pythium irregule;nannochloropsis sp.; and nitzschia sp.

Details regarding Grateloupia turuturu are disclosed in the articleentitled, “Grateloupia Turuturu (Halymeniaceae, Rhodophyta) is theCorrect Name of the Non-Native Species in the Atlantic Known asGrateloupia Doryphora,” Eur. J. Phycol. (2002), 37: 349-359, as authoredby Brigitte Gavio and Suzanne Fredericq, the disclosure which isincorporated by reference in its entirety.

Porphyridium cruentum is a red algae in the family porphyridiophyceaeand also termed rhodophyta and is used as a source for fatty acids,lipids, cell-wall polysaccharides and pigments. The polysaccharides ofthis species are sulphated. Some Porphyridium cruentum biomass containscarbohydrates of up to 57%.

Monodus subterraneus is described in an article entitled, “Biosynthesisof Eicosapentaenoic Acid (EPA) in the Fresh Water EustigmatophyteMonodus Subterraneus (Eustigmatophyceae),” J. Phycol, 38, 745-756(2002), authored by Goldberg, Shayakhmetova, and Cohen, the disclosurewhich is incorporated by reference in its entirety. The biosynthesis ofPUFAs from algae is complicated and the biosynthesis from this algae isdescribed in that article.

Phaeodactylum tricornutum is a diatom and unlike most diatoms, it cangrow in the absence of silicon and the biogenesis of silicifiedfrustules is facultative.

Isochrysis galbana is a microalgae and used in the bivalve aquacultureindustry.

Navicula sp. is a boat-shaped algae and is a diatom. Pythium irregule isa soilborne pathogen found on plant hosts.

Nannochloropsis sp. occurs in a marine environment, but also occurs infresh and brackish water. The species are small, nonmotile spheres thatdo not express any distinct morphological feature. These algae havechlorophyll A and lack chlorophyll B and C. They can build highconcentrations of pigment such as astaxanthin, zeaxanthin andcanthaxinthin. They are about 2-3 micrometers in diameter. They mayaccumulate high levels of polyunsaturated fatty acids.

Nitzschia sp. is a pinnate marine diatom and usually found in colderwaters and associated with both Arctic and Antarctic polar sea ice whereit is a dominant diatom. It produces a neurotoxin known as domoic acidwhich is responsible for amnesic shell fish poisoning. It may growexponentially at temperatures between −4 and −6 degrees C. It may beprocessed to form and extrapolate the fatty acids.

As a source of polyunsaturated fatty acids, microalgae competes withother micro-organisms such as fungi and bacteria. There may be somebacterial strains that could be an EPA source, but microalgae has beenfound to be a more adequate and readily available source. Microalgae isa good source of oil and EPA when derived from phaeodactylum, isochrysisand monodus. The microalgae Phaeodactylum tricornutum produces a highproportion of EPA. Other different strains and species of microalgae,fungi and possibly bacteria that can be used to source EPA include thefollowing:

I. Diatoms

Asterionella japonica

Bidulphia sinensis

Chaetoceros septentrionale

Lauderia borealis

Navicula biskanteri

Navicula laevis (heterotrof.)

Navicula laevis

Navicula incerta

Stauroneis amphioxys

Navicula pellicuolsa

Bidulphia aortia

Nitzschia alba

Nitzschia chosterium

Phaeodactylum tricornutum

Phaeodactylum tricornutum

Skeletonema costatum

II. Chrysophyceae

Pseudopedinella sp.

Cricosphaera elongate

III. Eustigmatophyceae

Monodus subterraneus

Nannochloropsis

IV. Prymnesiophyceae

Rodela violacea 115.79

Porphyry. Cruentum 1380.Id

V. Prasinophyceae

Pavlova salina

VI. Dinophyceae

Cochlodinium heteroloblatum

Cryptecodinium cohnii

Gonyaulax catenella

Gyrodinium cohnii

Prorocentrum minimum

VII. Other Microalgae

Chlorella minutissima

Isochrysis galbana ALII4

Phaeodactylum tricornutum WT

Porphyridium cruentum

Monodus subterraneus

VIII. Fungi

Mortierella alpine

Mortierella alpine IS-4

Pythium irregulare

IX. Bacteria

SCRC-2738

Different microalgae may be used to form the algae based oil comprisingglycolipids and phospholipids and at least EPA and/or EPA/DHA. Examplesinclude: Chlorophyta, Cyanophyta (Cyanobacteria), and Heterokontophyta.The microalgae may be from one of the following classes:Bacillariophyceae, Eustigmatophyceae, and Chrysophyceae. The microalgaemay be from one of the following genera: Nannochloropsis, Chlorella,Dunaliella, Scenedesmus, Selenastrum, Oscillatoria, Phormidium,Spirulina, Amphora, and Ochromonas.

Other non-limiting examples of microalgae species that may be usedinclude: Achnanthes orientalis, Agmenellum spp., Amphiprora hyaline,Amphora coffeiformis, Amphora coffeiformis var. linea, Amphoracoffeiformis var. punctata, Amphora coffeiformis var. taylori, Amphoracoffeiformis var. tennis, Amphora delicatissima, Amphora delicatissimavar. capitata, Amphora sp., Anabaena, Ankistrodesmus, Ankistrodesmusfalcatus, Boekelovia hooglandii, Borodinella sp., Botryococcus braunii,Botryococcus sudeticus, Bracteococcus minor, Bracteococcusmedionucleatus, Carteria, Chaetoceros gracilis, Chaetoceros muelleri,Chaetoceros muelleri var. subsalsum, Chaetoceros sp., Chlamydomasperigranulata, Chlorella anitrata, Chlorella antarctica, Chlorellaaureoviridis, Chlorella candida, Chlorella capsulate, Chlorelladesiccate, Chlorella ellipsoidea, Chlorella emersonii, Chlorella fusca,Chlorella fusca var. vacuolata, Chlorella glucotropha, Chlorellainfusionum, Chlorella infusionum var. actophila, Chlorella infusionumvar. auxenophila, Chlorella kessleri, Chlorella lobophora, Chlorellaluteoviridis, Chlorella luteoviridis var. aureoviridis, Chlorellaluteoviridis var. lutescens, Chlorella miniata, Chlorella minutissima,Chlorella mutabilis, Chlorella nocturna, Chlorella ovalis, Chlorellaparva, Chlorella photophila, Chlorella pringsheimii, Chlorellaprotothecoides, Chlorella protothecoides var. acidicola, Chlorellaregularis, Chlorella regularis var. minima, Chlorella regularis var.umbricata, Chlorella reisiglii, Chlorella saccharophila, Chlorellasaccharophila var. ellipsoidea, Chlorella salina, Chlorella simplex,Chlorella sorokiniana, Chlorella sp., Chlorella sphaerica, Chlorellastigmatophora, Chlorella vanniellii, Chlorella vulgaris, Chlorellavulgaris fo. tertia, Chlorella vulgaris var. autotrophica, Chlorellavulgaris var. viridis, Chlorella vulgaris var. vulgaris, Chlorellavulgaris var. vulgaris fo. tertia, Chlorella vulgaris var. vulgaris fo.viridis, Chlorella xanthella, Chlorella zofingiensis, Chlorellatrebouxioides, Chlorella vulgaris, Chlorococcum infusionum, Chlorococcumsp., Chlorogonium, Chroomonas sp., Chrysosphaera sp., Cricosphaera sp.,Crypthecodinium cohnii, Cryptomonas sp., Cyclotella cryptica, Cyclotellameneghiniana, Cyclotella sp., Dunaliella sp., Dunaliella bardawil,Dunaliella bioculata, Dunaliella granulate, Dunaliella maritime,Dunaliella minuta, Dunaliella parva, Dunaliella peircei, Dunaliellaprimolecta, Dunaliella salina, Dunaliella terricola, Dunaliellatertiolecta, Dunaliella viridis, Dunaliella tertiolecta, Eremosphaeraviridis, Eremosphaera sp., Effipsoidon sp., Euglena spp., Franceia sp.,Fragilaria crotonensis, Fragilaria sp., Gleocapsa sp., Gloeothamnionsp., Haematococcus pluvialis, Hymenomonas sp., Isochrysis aff. galbana,Isochrysis galbana, Lepocinclis, Micractinium, Micractinium,Monoraphidium minutum, Monoraphidium sp., Nannochloris sp.,Nannochloropsis salina, Nannochloropsis sp., Navicula acceptata,Navicula biskanterae, Navicula pseudotenelloides, Navicula pelliculosa,Navicula saprophila, Navicula sp., Nephrochloris sp., Nephroselmis sp.,Nitschia communis, Nitzschia alexandrine, Nitzschia closterium,Nitzschia communis, Nitzschia dissipata, Nitzschia frustulum, Nitzschiahantzschiana, Nitzschia inconspicua, Nitzschia intermedia, Nitzschiamicrocephala, Nitzschia pusilla, Nitzschia pusilla elliptica, Nitzschiapusilla monoensis, Nitzschia quadrangular, Nitzschia sp., Ochromonassp., Oocystis parva, Oocystis pusilla, Oocystis sp., Oscillatorialimnetica, Oscillatoria sp., Oscillatoria subbrevis, Parachlorellakessleri, Pascheria acidophila, Pavlova sp., Phaeodactylum tricomutum,Phagus, Phormidium, Platymonas sp., Pleurochrysis carterae,Pleurochrysis dentate, Pleurochrysis sp., Prototheca wickerhamii,Prototheca stagnora, Prototheca portoricensis, Prototheca moriformis,Prototheca zopfii, Pseudochlorella aquatica, Pyramimonas sp.,Pyrobotrys, Rhodococcus opacus, Sarcinoid chrysophyte, Scenedesmusarmatus, Schizochytrium, Spirogyra, Spirulina platensis, Stichococcussp., Synechococcus sp., Synechocystisf, Tagetes erecta, Tagetes patula,Tetraedron, Tetraselmis sp., Tetraselmis suecica, Thalassiosiraweissflogii, and Viridiella fridericiana. Preferably, the microalgae areautotrophic.

It is also possible to form the oil comprising glycolipids andphospholipids and at least EPA from genetically modified yeast.Non-limiting examples of yeast that can be used include Cryptococcuscurvatus, Cryptococcus terricolus, Lipomyces starkeyi, Lipomyceslipofer, Endomycopsis vernalis, Rhodotorula glutinis, Rhodotorulagracilis, Candida 107, Saccharomyces paradoxus, Saccharomyces mikatae,Saccharomyces bayanus, Saccharomyces cerevisiae, any Cryptococcus, C.neoformans, C. bogoriensis, Yarrowia lipolytica, Apiotrichum curvatum,T. bombicola, T. apicola, T. petrophilum, C. tropicalis, C. lipolytica,and Candida albicans. It is even possible to use a biomass as a wildtype or genetically modified fungus. Non-limiting examples of fungi thatmay be used include Mortierella, Mortierrla vinacea, Mortierella alpine,Pythium debaryanum, Mucor circinelloides, Aspergillus ochraceus,Aspergillus terreus, Pennicillium iilacinum, Hensenulo, Chaetomium,Cladosporium, Malbranchea, Rhizopus, and Pythium.

It is also possible that bacteria may be used that includes lipids,proteins, and carbohydrates, whether naturally occurring or by geneticengineering. Non-limiting examples of bacteria include: Escherichiacoli, Acinetobacter sp. any actinomycete, Mycobacterium tuberculosis,any streptomycete, Acinetobacter calcoaceticus, P. aeruginosa,Pseudomonas sp., R. erythropolis, N. erthopolis, Mycobacterium sp., B.,U. zeae, U. maydis, B. lichenformis, S. marcescens, P. fluorescens, B.subtilis, B. brevis, B. polmyma, C. lepus, N. erthropolis, T.thiooxidans, D. polymorphis, P. aeruginosa and Rhodococcus opacus.

Possible algae sourced, EPA/DHA based oils that are derived from analgae and contain glycol and phospholipid bound EPA and/or EPA/DHA andmay include a significant amount of free fatty acids, triglycerides andphospholipids and glycolipids in the range of 35-40% or more of totallipids are disclosed in the treatise “Chemicals from Microalgae” asedited by Zvi Cohen, CRC Press, 1999. Reference is also made to a studyin men that have been given a single dose of oil from a polar-lipid richoil from the algae Nannochloropis oculata as a source of EPA anddescribed in the article entitled, “Acute Appearance of Fatty Acids inHuman Plasma—A Comparative Study Between Polar-Lipid Rich Oil from theMicroalgae Nannochloropis Oculata in Krill Oil in Healthy Young Males,”as published in Lipids in Health and Disease, 2013, 12:102 by Kagan etal. The EPA in that algae oil was higher than that of krill oil by about25.06 to 13.63 for fatty acid composition as the percent of oil. Thealgae oil was provided at 1.5 grams of EPA and no DHA as compared tokrill oil that was provided at 1.02 grams EPA and 0.54 grams DHA. Theparticipants consumed both oils in random order and separated by sevendays and the blood samples were collected before breakfast and atseveral time points up to 10 hours after taking the oils.

The researchers determined that the algae based oil had a greaterconcentration of EPA and plasma than krill oil with the EPAconcentration higher with the algae based oil at 5, 6, 8 and 10 hours(P<0.05) intended to be higher at 4 hours (P=0.094). The maximumconcentration (CMAX) of EPA was higher with algae oil than with krilloil (P=0.010). The maximum change in concentration of EPA from itsfasting concentration was higher than with krill oil (P=0.006). The areaunder the concentration curve (AUC) and the incremental AUC (IAUC) wasgreater (P=0.020 and P=0.006). This difference may relate to thedifferent chemical composition and possibly the presence of theglycolipids where the presence of DHA in krill oil limits theincorporation of EPA into plasma lipids. Also, the n-3 polyunsaturatedfatty acids within glycolipids as found in the algae oil, but not in akrill oil, may be an effective system for delivering EPA to humans.

Microalgae can be cultured photoautotrophically outdoors to prepareconcentrated microalgae products containing Eicosapentaenoic acid (EPA)and Docosahexaenoic acid (DHA), which are the long-chain polyunsaturatedfatty acids (PUFAs) found in fish oil. Both are very important for humanand animal health. The concentrated microalgae products as disclosed inthe '037 patent may contain EPA and DHA and lipid products containingEPA and DHA purified from microalgae. The concentrated microalgaecomposition may be prepared by cultivating microalgaephotoautotrophically outdoors in open ponds under filtered sunlight in acontinuous or batch mode and at a dilution rate of less than 35% perday. The microalgae may be harvested in the exponential phase when thecell number is increasing at a rate of at least 20% of maximal rate. Inone example, the microalgae is concentrated. In another example, atleast 40% by weight of lipids in the microalgae are in the form ofglycodiacylglycerides, phosphodiacylglycerides, or a combination thereofand at least 5% by weight of the fatty acids are DHA, EPA, or acombination thereof.

In one example, the microalgae are Tetraselmis sp. cultivated at above20° C. or in another example at above 30° C. The EPA yield in themicroalgae has been found to be at least 10 mg/liter culture. Themicroalgae can be Isochrvsis sp. or Pavlova sp. in another example, orare Thalassiosira sp. or Chaetecoros sp. The microalgae may be differentdiatoms and are cultivated photoautotrophically outdoors in open pondsfor at least 14 days under filtered sunlight and at least 20% by weightof the fatty acids are EPA.

The use of this algae based oil overcomes the technical problemsassociated with the dwindling supplies of fish oil and/or Antarctickrill, which are now more difficult to harvest and obtain and useeconomically because these products are in high demand. A majordifference between fish oils and algae based oils is their structure.Fish oils are storage lipids and are in the form of triacylglycerides.The algae based oils as lipids are a mixture of storage lipids andmembrane lipids. The EPA and DHA present in algae based oils is mainlyin the form of glycolipids and a small percentage is in the form ofphospholipids. Glycolipids are primarily part of chloroplast membranesand phospholipids are part of cell membranes.

The '037 patent describes various methods for culturing microalgaephotoautotrophically outdoors to produce EPA and DHA. One method used isfiltering sunlight to reduce the light intensity on the photoautotrophicculture. Shade cloth or netting can be used for this purpose. It wasdetermined that for most strains, the optimal solar intensity forgrowth, for maintaining a pure culture, and for omega-3 fatty acidaccumulation was about 40,000 to 50,000 lux, approximately half of the110,000 lux of full sunlight. Shade cloth or netting is suitable forfiltering the sunlight to the desired intensity.

It is also possible to culture microalgae photoautotrophically outdoorsand produce EPA and DHA by using small dilutions and a slow dilutionrate of less than 40% per day, preferably less than 35% per day, morepreferably from about 15% to about 30% per day. In other examples, thedilution rate is 15-40% per day or 15-35% per day, and in yet otherexamples, the dilution rate is 10-30%, 10-35%, or 10-40% per day. Thesesmaller dilutions and lower dilution rates than are usually used helpprevent contamination in outdoor photoautotrophic cultures. It alsopromotes thick culture growth that gives good DHA or EPA yield.

Another technique to successfully culture microalgaephotoautotrophically outdoors and produce EPA and EPA/DHA is to harvestthe microalgae in exponential phase rather than stationary phase.Harvesting in exponential phase reduces the risk of contamination inoutdoor photoautotrophic cultures and has surprisingly been found togive a good yield of EPA and DHA. To drive fat accumulation in microbialcultures, the cultures are harvested in stationary phase because cellsin the stationary phase tend to accumulate storage lipids. The '037patent teaches that EPA and DHA accumulate in large amounts as membranelipids in cultures harvested in the exponential phase. The membranelipids containing EPA and DHA are predominantly phosphodiacylglyceridesand glycodiacylglycerides, rather than the triaclyglycerides found instorage lipids. These cultures are harvested often when cell number isincreasing at a rate at least 20% of the maximal rate, i.e., the maximalrate achieved at any stage during the outdoor photoautotrophic growth ofthe harvested culture. In specific examples, the cultures are harvestedin exponential phase when cell number is increasing at a rate of atleast 30%, at least 40%, or at least 50% of maximal rate. It is alsopossible to use recombinant DNA techniques.

The '037 patent includes several examples, which are referenced to thereader for description and teaching purposes.

Example 1

The strain Thalassiosira sp. is a diatom and this strain used wasisolated from Bay of Bengal, and it dominates during summer months. Thisexample strain was isolated from seawater collected near Chemai, India,and the culture was maintained in open tubs. The particular strain wasidentified as Thalassiosira weissflogii, which is capable of growth athigh temperatures (35-38° C.). The fatty acid profile was good even whenthe alga was grown at high temperature with 25-30% EPA (as a percentageof fatty acids).

Culturing:

The lab cultures were maintained in tubs in an artificial seawatermedium, under fluorescent lights (3000-4000 lux) and the temperature wasmaintained at 25° C. Initial expansion of the culture was done underlaboratory condition in tubs. The dilution rate was 15% to 30% of thetotal culture volume per day. Once the volume was 40-50 liters, it wastransferred to an outdoor pond. The outdoor ponds were covered withnetting to control the light (40,000 to 50,000 lux). The dilutioncontinued until the culture reached 100,000 liters volume. The culturewas held in 500 square meter ponds at this time with a culture depth of20 cm. The culture was stirred with a paddle wheel and CO2 was mixed tokeep the culture pH neutral. When the EPA levels in the pond reached adesirable level (10-15 mg/lit), the whole pond was harvested byfiltration. The filtered biomass was washed with saltwater (15 parts perthousand concentration) and then spray dried. The mode of culturing wasbatch mode. The EPA productivity was 2-3 mg/lit/day. The ponds can alsobe run continuously for several weeks by harvesting part of the culture,recycling the filtrate into the ponds and replenishing requirednutrients.

Example 2

The strain Tetraselmis sp. is in the division Chlorophyta and the classProsinophyceae or Micromanadophyceae. This strain was obtained from theCentral Marine Fisheries Research Institute, India. It was isolated fromthe local marine habitats in India. The culture was maintained in flasksin artificial seawater medium, and expanded as described forThalassiosira. With culture outdoors in open ponds as described forThalassiosira, the strain gave a good lipid yield (200-300 mg/liter) andan EPA content of 6-7% of fatty acids.

Example 3

The strain Chaetoceros sp. is another diatom strain obtained from theCentral Marine Fisheries Research Institute, India, and isolated fromlocal marine habitats in India. Chaetoceros sp. was maintained in flasksand cultivated in outdoor ponds photoautotrophically as described inExample 1. It gave similar EPA productivity and EPA content asThalassiosira as described in Example 1.

Example 4

The strain Isochrysis sp. is in the Prymnesiophyta, classPrymnesiophyceae, order Isochrysidales. It was obtained from the CentralMarine Fisheries Research Institute, India, and isolated from localmarine habitats in India. It was maintained and grown as described inExample 1. It was expanded from laboratory culture to a 50,000 literoutdoor pond culture in 14-15 days with a dilution rate of 15-30% perday. The lipid content at harvest was 100-150 mg lipids/liter. The rateof lipid production was 25-50 mg/liter/day. DHA was 10-12% of totalfatty acids.

Example 5

Harvesting and Drying: The harvesting may be done by flocculation. Thecommonly used flocculants include Alum with polymer and FeCl3 with orwithout polymer and chitosan. The concentration of flocculent willdepend on the cell number in the culture before harvest. The range mayvary from 100 ppm to 500 ppm. Alternatively, harvesting is done byfiltration using appropriate meshes. Removal of adhered chemicals (otherthan salt) is accomplished by washing the cells in low salinity water.

The harvested slurry is then taken for spray drying. The slurry issometimes encapsulated to prevent oxidation. The concentration ofencapsulating agent may vary from 0.1 to 1.0% on a dry weight basis.Modified starch is a suitable encapsulating agent. The spray dryer isusually an atomizer or nozzle type. The inlet temperature ranges from160 to 190° C. and the outlet temperature ranges from 60 to 90° C. Thespray dried powder is used immediately for extraction. If storage isrequired, the powder is packed in aluminum laminated pouches and sealedafter displacing the air by nitrogen. The packed powder is stored atambient temperature until further use.

Example 6

Extraction of EPA/DHA is carried out using a wet slurry or dry powderand solvents, which include hexane, ethanol, methanol, acetone, ethylacetate, isopropanol and cyclohexane and water, either alone or incombination of two solvents. The solvent to biomass ratio depends on thestarting material. If it is a slurry, the ratio is 1:2 to 1:10. With aspray dried powder, on the other hand, the ratio is 1:4 to 1:30. Theextraction is carried out in an extraction vessel under inertatmosphere, with temperature ranges from 25 to 60° C. and with timevarying from one hour to 10 hours. Solvent addition is made one time orin parts based on the lipid level in the cells.

After extraction of crude lipid, the mixture is passed through acentrifuge or filtration system to remove the cell debris. The lipid inthe filtrate is concentrated by removing the solvent by distillation,which is carried out under vacuum. The resulting product is a crudelipid extract, which contains approximately 10% omega-3 fatty acid(EPA/DHA). The extract can be used as it is or purified further toenrich the omega-3 fatty acids. Further purification may involve removalof unsaponifiables such as pigments, sterols and their esters. The algaebased oil composition may be used for different purposes as described.

In the incorporated by reference '072 and '608 patents, a clinical trialusing astaxanthin alone is described where a dosage of one softgelcontaining 15 milligrams of astaxanthin was given once a day duringbreakfast for 12 weeks and 70 subjects recruited for the study. This wasa comparative single blind clinical trial and a total of 70 subjectsrecruited for the study with 35 in each group corresponding to anastaxanthin oleoresin complex and a placebo-control. The clinical trialresults are reproduced below and show the efficacy of using high dosagesof astaxanthin at levels of 15 mg. It has been found, however, thatsurprisingly effective results are used when 2 to 4 mg or 0.5 to 12 mgor other ranges as described of astaxanthin are used alone in thepresence of an adequate surfactant such as a sunflower or perilla basedphospholipid. This could include the roe extract with phospholipid asdescribed above. This could be a plant based phospholipid also and alecithin alone or modified as a lysophospholipid source. It is possibleto use glycophospholipids. An example perilla oil is described anddisclosed in the incorporated by reference and commonly assigned '904patent. The astaxanthin and surfactant may optionally be admixed withlow molecular weight hyaluronic acid as described above or UC-II. Theastaxanthin in the presence of a surfactant may be at below 4 mg/day andas noted before, optionally admixed with the low molecular weighthyaluronic acid or UC-II and/or as a chicken sternum collagen isolate.The phospholipid may have little EPA and DHA. In one example, apreferred astaxanthin concentration is about 2-4 mg and a chickensternum collagen isolate can be about 40 mg and have a range of 30 toabout 50 mg. Other surfactants such as plant based phospholipids andcommercially available lecithins that are modified and including eggyolk compositions and/or sea based oils such as from perilla may beused. Sea based phospholipids and lysolipid, also referred to aslysophospholipid, counterparts may be used. A non-omega-3 platform maybe used with the current invention. The low molecular weight hyaluronicacid as described may vary from 1-500 mg, 10-70 mg, 35 mg, or 45 mg, andother ranges as described, and is a preferred low molecular weightmicrobial fermented product as described above.

The clinical trial as set forth in the '072 and '608 patents is now setforth.

Clinical Trial to Evaluate the Efficacy of Haematococcus pluvialisAstaxanthin Oleoresin Complex in Osteoarthritis Patients:

The study has been carried out as a comparative single blind clinicaltrial of astaxanthin oleoresin complex in 60 Osteoarthritis patients ascompared with Placebo control for a period of 12 weeks n=60 (30 A+30 P).The dosage consisted of one softgel containing 15 mg of Astaxanthin oncea day during breakfast for 12 weeks. A total of 70 subjects wererecruited for the study, 35 in each group (Astaxanthin oleoresin complexand placebo-control) of both the sexes. Patients were explained thenature of the study and informed consent was obtained prior to the startof the study. Patient subjects were clinically examined by the PrincipalInvestigator and team. X ray and blood samples were drawn at thecommencement and at the end of study period. The case record forms werefilled by the Principal Investigator and rechecked by the Clinicalresearch associate. Sixty patient subjects completed the study. Ten weredrop outs due to various reasons but not on account of intolerance tothe astaxanthin oleoresin complex or placebo control. The results weretabulated by the expert data entry operators under supervision ofBiometric expert. The results were subjected to Statistical analysis byan independent analyst.

The assessment of Osteoarthritis symptoms were based on Western Ontarioand McMasters Universities (WOMAC) Osteoarthritis Index, VAS scale,Lequesne's functional scale as well as Sleep score as additionalparameters besides radiological investigations. Further the assessmentof Osteoarthritis symptoms based on haematological studies, specificallyMMP3 (Matrix metalloproteinase 3) in clinical parameters sinceOsteoarthritis patients show elevated levels of MMP3 in blood as well asin synovial fluid. The elevated levels cause significant tissue damagethrough cartilage destruction.

Results of Clinical Trial and Discussions: Total Health AssessmentScore—

The total health assessment on Osteoarthritis patients was carried outon their difficulty to a) Dressing—doing buttons, washing and combinghair; b) Arising—stand up straight from a chair, get in and out of bed,sit cross-legged on floor and get up; c) Eating—cut vegetables, lift afull cup/glass to your mouth; d) Walking—walk outdoor on flat ground,climb up five steps; and e) Hygiene—Take a bath, wash and dry your body,get on and off the toilet; f) Reaching—reach and get down a 2 kg objectfrom just above your head, bend down to pick up clothing from the floor;g) Grip-open a bottle previously opened, turn taps on & off, open doorlatches; h) Activities—work in office/house, run errand to shop, get inand out of car/auto. The summary of results is given Table 3.

There were significant reductions in the mean scores of patients takingastaxanthin oleoresin complex at the end of 3 months but not for thePlacebo group. There were no significant differences between astaxanthinand Placebo group at Basal values. There were significant differencesbetween the astaxanthin and placebo group at 3 months.

WOMAC Score—

The Western Ontario McMaster (WOMAC) is a validated instrument designedspecifically for the assessment of lower extremity pain and function inOsteoarthritis (OA) of the knee. The patients were assessed on theirpain, stiffness and difficulty in carrying out day-to-day activities.The pain index was assessed for Activities—a) in walking on flatsurface, going up or down on flat surface, at night while in bed,sitting or lying, standing upright; b) Stiffness—after first wakening inmorning, after sitting/lying or resting later in the day; and c)difficulty in descending stairs, ascending stairs, standing up from achair, while standing, bending to floor to pick up objects, walking onflat ground, getting in and out of autorickshaw/bus/car, going shopping,on rising from bed, while lying on bed, while sitting on chair, goingon/off toilet, doing heavy domestic duties such as moving heavyboxes/scrubbing floor/lifting shopping bags, doing light domestic dutiessuch as cleaning room/table/cooking/dusting, while sitting cross-leggedposition, rising from cross-legged position, while squatting on floor.The summary of the results are given in Table 4.

There were significant reductions in the mean scores for patients takingAstaxanthin oleoresin complex at the end of 3 months but not for thePlacebo group. There were no significant differences between patientstaking Astaxanthin oleoresin complex and placebo group at basal values.There were significant differences between Astaxanthin and Placebogroups at 3 months.

VAS (Visual Analog Scale) on Pain Parameters—

Pain parameters were assessed in Osteoarthritis patients takingastaxanthin oleoresin and the Placebo group using VAS. The assessmentwas carried out in a) Pain parameters—pain while using stairs, painwhile walking on flat ground, pain while standing upright, pain whilesitting or lying down, pain at night in bed b) Physical functions—goingdownstairs, going upstairs, sitting, getting up from sitting, standing,bending to floor, walking on flat ground, getting into or out ofautomobiles, shopping, putting on socks/stockings, taking offsocks/stockings, getting into bed, getting out of bed, getting into orout of bath tub, getting on or off toilet seat, during heavy householdchores, during light household chores, getting into lotus position. Thesummary of results of Pain parameters (Pain+Physical) scores are givenin Table 5.

There were significant reductions in the mean scores at the end of 3months for patients taking Astaxanthin oleoresin complex but not for thePlacebo group. There were no significant differences between Astaxanthinoleoresin complex and Placebo group at Basal values. There weresignificant differences between Astaxanthin oleoresin complex andPlacebo groups at 3 months.

Laquesne's Index—

Laquesne's index is the Functional index for Osteoarthritis of the knee.Assessment is carried out on a) Pain/discomfort—during nocturnal bedrest, morning stiffness or regressive pain after rising, after standingfor 30 minutes; and b) Physical functions—maximum distance walked,activities of daily living like able to climb up a standard flight ofstairs, able to climb down a standard flight of stairs, able to squat orbend on the knees, able to walk on uneven ground. The Laquesne's indexresults are given in Table 6.

There were significant reductions in mean scores for the patients takingAstaxanthin oleoresin complex at the end of 3 months but not for thePlacebo group. There were no significant differences between astaxanthinoleoresin complex and Placebo groups at Basal values. There weresignificant differences between astaxanthin oleoresin complex andPlacebo groups at 3 months.

Sleep Scale—

Sleep is an important element of functioning and well being. Sleep Scalewas originally developed in the Medical Outcomes Study (MOS) intended toassess the extent of sleep problems. The Medical Outcomes Study SleepScale includes 12 items assessing sleep disturbance, sleep adequacy,somnolence, quantity of sleep, snoring, and awakening short of breath orwith a headache. A sleep problems index, grouping items from each of theformer domains, is also available. This assessment evaluated thepsychometric properties of MOS-Sleep Scale in Osteoarthritis patientstaking Astaxanthin oleoresin complex and Placebo group. The results onSleep scale MOS is given in Table 7.

There were significant reductions in the mean scores for patients takingastaxanthin oleoresin complex at the end of 3 months but not for thePlacebo group. There were no significant differences between astaxanthinoleoresin complex group and Placebo group at Basal values. There weresignificant differences between astaxanthin oleoresin complex group andPlacebo group for most of the variables.

MMP3 (Matrix Metalloproteinase 3) Assay—

Assessment of Osteoarthritis symptoms based on haematological studies,specifically MMP3 (Matrix metalloproteinase 3) were carried out inclinical parameters since Osteoarthritis patients show elevated levelsof MMP3 in blood as well as in synovial fluid. The elevated levels causesignificant tissue damage through cartilage destruction. The results ofthe MMP3 analysis on Osteoarthritis patients before and after 3 monthsof administering with astaxanthin oleoresin complex are given in FIG. 2.The results of the MMP3 analysis on Osteoarthritis patients before andafter 3 months of administering with Placebo are given in FIG. 3. MMP3levels did not show significant change but the trend is towardsreduction.

In all, 70 subjects were recruited for the study in a randomized manner.The patients were explained the nature of the study as well as active(astaxanthin oleoresin complex softgels containing 15 mg astaxanthin)and placebo treatments. An informed written consent was obtained fromthe subjects prior to the commencement of the study. At the commencementof the study patient subjects were clinically examined and blood sampleswere collected for CBC/ESR & MMP3 study. Specific orthopaedic andradiological examinations were performed. The patient subjects wereassigned placebo and active treatment in a random manner for a period of12 weeks. Patient subjects were advised to continue with their otherroutine treatments, if any. At the end of 4 weeks the subjects werecalled for a second visit in order to refill the samples. The sameprocedure was carried out in third visit and the procedure of the firstvisit was repeated in fourth visit. Results were tabulated by data entryoperators and detailed statistical analysis was performed using thoseresults. At the base level the groups were similar and comparable.

Advantages of the Invention

Total Health Assessment score (Arising, Dressing, Eating, Walking,Hygiene, Grip, Reaching, Daily activities) exhibited significant changesbetween Astaxanthin oleoresin complex and Placebo group (P<0.001).Improvement was seen in all the parameters of daily activities.

WOMAC INDEX exhibited significant differences (P<0.001). This score isunique for the functional abilities in patients with chronic jointdisorders such as Osteoarthritis.

VAS Pain parameters (Pain+Physical) score: There were significantreductions in the mean scores at the end of treatment for patientstaking astaxanthin oleoresin complex but not for Placebo P (<0.001). Itis suggestive of improvement in the pain related aspects ofOsteoarthritis.

Laquesne's index: (Functional Index for OA of knee): There weresignificant reductions in the mean scores at the end of treatment forpatients taking Astaxanthin oleoresin complex but not for Placebo(P<0.05).

Sleep scale from the medical outcomes study: There were significantreductions in the mean scores at the end of treatment for patientstaking astaxanthin oleoresin complex but not for Placebo (P<0.001).

There was significant difference between the average sleep each night(hrs). Patients taking astaxanthin oleoresin complex had higher sleepthan Placebo group (P<0.01).

Improvement in the sleep time clearly indicates efficacy of thetreatment with astaxanthin oleoresin complex. Astaxanthin helps to getbetter sleep as is evident from sleep score. This is due to reduction inpain and other symptoms of the disorder MMP3 did not show significantchange but the trend is towards reduction. Reduction in MMP3 levels aresuggestive of improving cartilage health due to reduction in the processof cartilage destruction in a positive manner although there is neitherdirect proof to this effect nor statistically significant effect in thepresent study. No change in the radiological picture was seen. Nonoteworthy side effect/intolerance was noted during the study period.Astaxanthin oleoresin complex appears to be safe for generalconsumption.

Astaxanthin oleoresin complex extracted through polar solvents fromHaematococcus pluvialis alga may be suitable for the patients in theearly stage of the Osteoarthritis to prevent the progression of thedisorder. It may be useful to the patients with establishedOsteoarthritis to provide symptomatic relief from pain and improvedquality of life. Astaxanthin oleoresin complex improves symptoms likepain as well as quality of physical activities of daily life in asignificant manner. Osteoarthritis is seen to mark its presence at ayounger age in India. It would be appropriate to initiate the treatmentwith Astaxanthin oleoresin complex right from the beginning as soon asthe diagnosis is arrived at. Study with larger sample size at differentcentres is recommended to study the mechanism of action of Astaxanthinoleoresin complex in Osteoarthritis further.

TABLE 1 Carotenoid Profile of Haematococcus Pluvialis Cell Powder andAstaxanthin Oleoresin Complex Astaxanthin Carotenoids Cell powderOleoresin complex 5% Beta-carotene 0.62 ± 0.01 0.62 ± 0.01 Canthaxanthin1.21 ± 0.03 1.20 ± 0.03 Astacene 3.09 ± 0.06 3.09 ± 0.06 Semiastacene1.35 ± 0.03 1.35 ± 0.03 Dicis astaxanthin 1.07 ± 0.02 1.03 ± 0.05 Transastaxanthin 75.70 ± 1.53  75.75 ± 1.51   9 cis astaxanthin 9.20 ± 0.779.19 ± 0.77 13 cis astaxanthin 6.10 ± 0.94 6.08 ± 0.93 Lutein 1.66 ±0.03 1.65 ± 0.03

TABLE 2 Proximate Analysis, Carotenoid Profile and Fatty Acid Profile ofAstaxanthin Oleoresin Complex Astaxanthin oleoresin PARAMETER complex 5%PHYSICAL Appearance Free flow Color Dark red PROXIMATE Protein % 0.95 ±0.03 Carbohydrate % 0.11 ± 0.01 Lipid % 94.89 ± 0.12  Ash % 3.82 ± 0.08Moisture % 0.23 ± 0.02 Carotenoids 5.14 ± 0.04 CAROTENOIDS % Totalcarotenoids 5 Total astaxanthin 4.68 [all-trans-astaxanthin [3.909-cis-astaxanthin 0.47 13-cis-astaxanthin 0.31 15-cis-astaxanthin 0Dicis - astaxanthin] 0.05] Betacarotene 0.03 Canthaxanthin 0.06 Lutein0.08 FATTY ACID PROFILE, Area % C4:0 Myristic acid 0.23 C15:0Pentadecanoic acid 0.1 C16:0 Palmitic acid 24.57 C16:1 Palmitoleic acid0.57 C16:2 Hexadeca dienoic acid 0.45 C16:3 Hexadecatrienoic acid 0.14C16:4 Hexadecatetraenoic acid 1.15 C17:0 Heptadecanoic acid 2.14 C18:0Stearic acid 1.61 C18:1 Oleic acid 38.93 C18:2 Linoleic acid 17.22C18:3, n-6 Gamma linolenic acid 0.84 C18:3, n-3 Alpha linolenic acid8.14 C18:4 Octadeca tetraenoic acid 1.3 C20:2 Eicosadienoic acid 0.81C20:4 Arachidonic acid 0.85 C22:0 Behenic acid 0.5

TABLE 3 Total Health Assessment Score Total Health Assessment ScoreDuration Significance Treatments Basal 1 month 2 months 3 months levelAstaxanthin 18 14.68 13.19 12.13 S, P < 0.001 Placebo 20.25 19.8 19.4819.51 NS, P = 0.4 S = Significant, NS = Not Significant, P = Probability

TABLE 4 WOMAC Score WOMAC Duration Significance Treatments Basal 1 month2 months 3 months level Astaxanthin 36.39 31.87 28.42 26.52 S, P < 0.001Placebo 38.07 36.62 36.59 36.1 NS, P = 0.6 S = Significant, NS = NotSignificant, P = Probability

TABLE 5 VAS Pain Parameters Score Pain Parameters Duration SignificanceTreatments Basal 1 month 2 months 3 months level Astaxanthin 891.94828.71 772.58 748.39 S, P < 0.001 Placebo 945.86 923.28 916.21 915.17NS, P = 0.1 S = Significant, NS = Not Significant, P = Probability

TABLE 6 Laquesne's Index Significance Parameters Astaxanthin Placebolevel 1. During nocturnal Basal 0.6 +/− 0.7 0.6 +/− 0.7 NS, P = 1.0 bedrest 3 0.8 +/− 0.7 0.5 +/− 0.7 S, P = 0.05 months 2. Morning stiffnessBasal 0.9 +/− 0.6 0.6 +/− 0.7 NS, P = 0.9 or regressive pain 3 0.6 +/−0.6 0.6 +/− 0.5 NS, P = 0.9 after rising months 3. After standing forBasal 0.4 +/− 0.5 0.6 +/− 0.7 NS, P = 0.9 30 minutes 3 0.3 +/− 0.6 0.5+/− 0.7 S, P = 0.05 months 4. Maximum Basal 1.3 +/− 0.7 1.7 +/− 1.3 NS,P = 0.9 distance walked 3 0.6 +/− 0.5 1.7 +/− 1.3 S, P = 0.001 months 5.Activities of daily living a) Able to climb up Basal 0.8 +/− 0.5 0.9 +/−0.3 NS, P = 0.9 a standard flight of 3 0.7 +/− 0.5 1.0 +/− 0.4 S, P =0.03 stairs months b) Able to climb Basal 1.3 +/− 0.3 1.6 +/− 0.9 NS, P= 0.9 down a standard 3 0.9 +/− 0.6 1.6 +/− 0.9 S, P = 0.03 flight ofstairs months c) Able to squat or Basal 1.3 +/− 0.3 1.6 +/− 0.9 NS, P =0.9 bend the knees 3 0.9 +/− 0.6 1.6 +/− 0.9 S, P = 0.03 months d) Ableto walk on Basal 1.3 +/− 0.3 1.6 +/− 0.9 NS, P = 0.9 uneven ground 3 0.9+/− 0.6 1.6 +/− 0.9 S, P = 0.03 months S = Significant, NS = NotSignificant, P = Probability

TABLE 7 Sleep Scale MOS Significance Sleep parameters AstaxanthinPlacebo level 1. Time to fall Basal 2.3 +/− 1.3 2.6 +/− 1.3 NS, P = 0.9asleep (min) 3 1.6 +/− 1.1 2.5 +/− 1.3 S, P < 0.001 during the past 4months weeks 2. Average sleep 6. +/− 1.3 5. +/− 1.8 S, P < 0.001 eachnight (hours during last 4 weeks) 3. Feel your Basal 3. +/− 1.9 3. +/−1.9 NS, P = 0.9 sleep was not 3 2.6 +/− 2.1 3.8 +/− 2.1 S, P = 0.02quiet? months 4. Get enough Basal 3.8 +/− 1.9 3.8 +/− 1.9 NS, P = 1.0.sleep to feel 3 2.9 +/− 2.1 3.6 +/− 2.1 S, P = 0.03 rested upon? months5. Awaken short Basal 5.6 +/− 1.2 4.6 +/− 2.2 NS, P = 0.6 of breath orwith 3 5.6 +/− 1.2 4.5 +/− 2.8 NS, P = 0.6 headache? months 6. Feeldrowsy or Basal 5. +/− 1.2 4. +/− 2.2 NS, P = 0.6 sleepy during 3 5.7+/− 1.8 4.5 +/− 2.8 NS, P = 0.6 day? months 7. Have trouble Basal 3.6+/− 2.7 4.6 +/− 2.2 NS, P = 0.3 falling asleep? 3 4.4 +/− 2.1 4.5 +/−2.8 NS, P = 0.9 months 8. Awaken Basal 4.1 +/− 2.9 4.6 +/− 2.2 NS, P =0.7 during your sleep 3 4.9 +/− 2.3 4.5 +/− 2.8 NS, P = 0.7 time andhave months trouble in falling sleep again? 9. Have trouble Basal 4.7+/− 1.8 4.6 +/− 2.2 NS, P = 0.9 staying awake 3 5.4 +/− 1.8 4.5 +/− 2.8S, P = 0.05 during the day? months 10. Snore during Basal 5.5 +/− 1.14.4 +/− 1.5 NS, P = 0.2 your sleep? 3 5.7 +/− 0.8 4.8 +/− 1.3 S, P =0.05 months 11. Take naps Basal 4.1 +/− 1.5 4.4 +/− 1.5 NS, P = 0.6 (5min. or longer) 3 3.7 +/− 1.5 4.8 +/− 1.3 S, P = 0.05 during the day?months 12. Get the Basal 3.2 +/− 1.8 4.5 +/− 1.5 NS, P = 0.6 amount ofsleep 3 3.7 +/− 1.8 4.8 +/− 1.3 S, P = 0.05 you needed? months S =Significant, NS = Not Significant, P = Probability

Many modifications and other embodiments of the invention will come tothe mind of one skilled in the art having the benefit of the teachingspresented in the foregoing descriptions and the associated drawings.Therefore, it is understood that the invention is not to be limited tothe specific embodiments disclosed, and that modifications andembodiments are intended to be included within the scope of the appendedclaims.

That which is claimed is:
 1. A dietary supplement composition formulatedin a therapeutic amount to treat and alleviate symptoms of joint pain ina patient, the dietary supplement composition comprisingpro-inflammatory low molecular weight microbial fermented sodiumhyaluronate fragments having a molecular weight of 0.5 to 300kilodaltons (kDa) in an oral dosage form.
 2. The dietary supplementcomposition according to claim 1, wherein the pro-inflammatory lowmolecular weight microbial fermented sodium hyaluronate fragments aremicro- or nano-dispersed within the composition.
 3. The dietarysupplement composition according to claim 1, wherein the dietarysupplement composition further comprises glucosamine.
 4. The dietarysupplement composition according to claim 3, wherein the glucosaminecomprises glucosamine hydrochloride (HCL).
 5. The dietary supplementcomposition according to claim 3, wherein the glucosamine comprisesglucosamine sulfate.
 6. The dietary supplement composition according toclaim 1, wherein the dietary supplement composition further comprisesany one of chondroitin, Boswellia serrata, turmeric,methylsulfonymethane (MSM), or s-adenosyl-methionine.
 7. The dietarysupplement composition according to claim 1, wherein the dietarysupplement composition further comprises Type II collagen.
 8. Thedietary supplement composition according to claim 1, wherein the dietarysupplement composition includes 10 to 70 mg of the pro-inflammatory lowmolecular weight microbial fermented sodium hyaluronate fragments havinga molecular weight of 0.5 to 300 kDa.
 9. The dietary supplementcomposition according to claim 1, wherein the dietary supplementcomposition further includes astaxanthin.
 10. A dietary supplementcomposition formulated in a therapeutic amount to treat and alleviatesymptoms of joint pain in a patient, the dietary supplement compositioncomprising 10 to 70 mg of pro-inflammatory low molecular weightmicrobial fermented sodium hyaluronate fragments having a molecularweight of 0.5 to 300 kilodaltons (kDa), wherein the dietary supplementcomposition is in the form of a single dosage capsule.
 11. The dietarysupplement composition according to claim 10, wherein thepro-inflammatory low molecular weight microbial fermented sodiumhyaluronate fragments are micro- or nano-dispersed within thecomposition.
 12. The dietary supplement composition according to claim10, wherein the dietary supplement composition further comprisesglucosamine.
 13. The dietary supplement composition according to claim12, wherein the glucosamine comprises glucosamine hydrochloride (HCL).14. The dietary supplement composition according to claim 12, whereinthe glucosamine comprises glucosamine sulfate.
 15. The dietarysupplement composition according to claim 12, wherein the dietarysupplement composition further comprises any one of chondroitin,Boswellia serrata, turmeric, methylsulfonymethane (MSM), ors-adenosyl-methionine.
 16. The dietary supplement composition accordingto claim 12, wherein the dietary supplement composition furthercomprises Type II collagen.
 17. The dietary supplement compositionaccording to claim 12, wherein the dietary supplement compositionfurther includes astaxanthin.